(Current Topics in Environmental Health and Preventive Medicine) Takemi Otsuki, Claudia Petrarca, Mario Di Gioacchino (Eds.)-Allergy and Immunotoxicology in Occupational Health-Springer Singapore (201

Current Topics in
Environmental Health and Preventive Medicine
Takemi Otsuki
Claudia Petrarca
Mario Di Gioacchino
Editors
Allergy and
Immunotoxicology
in Occupational
Health
Current Topics in
Environmental Health and Preventive Medicine
Series editor
T. Otsuki
Kurashiki, Japan
Current Topics in Environmental Health and Preventive Medicine, published in
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Takemi Otsuki Claudia Petrarca
Mario Di Gioacchino
Editors
Allergy and
Immunotoxicology in
Occupational Health
Editors
Takemi Otsuki Claudia Petrarca
Department of Hygiene Immuntotoxicology and Allergy Unit &
Kawasaki Medical School Occupational Biorepository, Center of
Kurashiki, Japan Excellence on Aging and Translational
Medicine (CeSI-MeT)
Mario Di Gioacchino G. DAnnunzio University Foundation
Immuntotoxicology and Allergy Unit & Chieti, Italy
Occupational Biorepository, Center of
Excellence on Aging and
Translational Medicine (CeSI-MeT)
G. DAnnunzio University Foundation
Chieti, Italy
Department of Medicine and Science
of Aging
G. dAnnunzio University
Chieti, Italy
ISSN 2364-8333 ISSN 2364-8341 (electronic)

Current Topics in Environmental Health and Preventive Medicine
ISBN 978-981-10-0349-3 ISBN 978-981-10-0351-6 (eBook)
DOI 10.1007/978-981-10-0351-6
Library of Congress Control Number: 2016953187
Springer Science+Business Media Singapore 2017

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Preface
This Book is published as a mid-term step (i.e., middle of every 3-year period) by
the Allergy and Immunotoxicology Scientific Committee (AISC) of the International
Congress of Occupational Health (ICOH). The comprehensive meeting of ICOH is
held every 3 years. Recently, it was held in Seoul, South Korea, 31 May5 June
2015, when Prof. Mario Di Gioacchino, of Chieti, Italy, served as the chairperson.
The next meeting will be held in Dublin, Ireland, in 2018. In conjunction with the
opening of the special, general, oral, and poster sessions as well as joint sessions
with other scientific committees (SCs), several SCs are staging mid-term activities
independently. Our SC/AISC has also been holding several special midterm sympo-
siums in Italy, China, and Japan (in Kumamoto and Kyoto) and calls each such
symposium an International Symposium on Occupational and Environmental
Allergy and Immune Diseases (ISOEAID). As the current chairperson of this SC,
Prof. Otsuki was the local organizer of ISOEAID 2010 held at Kyoto. It was April
and we were surrounded by beautiful cherry blossom trees in full bloom. During the
symposium, we presented and discussed many issues regarding the occupational
and environmental allergy and immunotoxicology field and enjoyed the lectures on
air pollution such as the effects of PM2.5 on immune organs, immunological aspects
of sick-building syndrome, sensitizers in GHS (The Globally Harmonized System
of Classification and Labelling of Chemicals), and childhood and immunology.
The first honorary chairperson in our SCs was Prof. Toshio Matsushita,
Kagoshima, Japan. Thereafter, Prof. Poalo Boscolo, Chieti, Italy, and Prof. Kanehisa
Morimoto, Osaka, Japan, inherited the chairpersons role. All three editors of this
eBook have been in that role in our SC. Prof. Di Gioacchino served as Chair 2009
2014. I have been the Chair since 2015, with Prof. Petrarca as the secretary in the
SCs. Subsequently we decided to publish this eBook instead of ISOEAID at this
mid-term point based on the presentation in the ICOH meeting in Seoul, South
Korea.
The AISC has been focusing on investigation and discussion of the pathophysio-
logical mechanisms involved with the allergic and immunotoxicological effects of
environmental as well as occupational substances. Particularly, occupational allergies
v
vi Preface
such as dermatitis and asthma were practically classical occupational diseases. The
basic strategy is to avoid exposure, whereas there have been numerous newly devel-
oped substances that are being introduced in occupational situations. Thus, establish-
ing methods of prediction using in vitro and in silico procedures is required. In
addition to these investigations, epidemiological studies are also important, as is
establishing a system to quickly identify allergic diseases caused by newer substances,
and it is valuable, as well, to prevent workers from contracting these diseases. In addi-
tion, with allergic diseases the immunotoxicological effects of various substances in
the environment as well as occupational settings are also important. Major members
of our SC have been investigating the immunotoxicological effects of fibrous and
particulate matters such as silica, asbestos, and recently nanomaterials including
nanoparticles, nanotubes, and nanosheets.
In the specialty session for allergy, the review of prevention and management of
allergens in occupational allergy, traditional and emerging occupational allergy in
Japan, review of work-related and non-industrial indoor-related asthma, and molec-
ular and cellular mechanisms in lung-specific immune responses activated by some
particulates were presented. Regarding immunotoxicology, epidemiological health
surveillance in formerly asbestos-exposed workers as well as experimental work
regarding the effects of asbestos on human regulatory T cells were presented. In
addition, understanding immunotoxicity of engineered nanomaterials for sustain-
able nanotechnology was also presented.
There were many poster presentations in Korea on glass allergic asthma, sensiti-
zation in workers exposed to urban air pollution, in silico analysis such as the quali-
tative structuretoxicity relationship (QSTR), solar radiation and the immune
system, and phagocytic alteration in leukocytes exposed to benzene were the field I
allergic insights. From the immunotoxicological aspect, many presentations regard-
ing effects of asbestos on various human immune cells such as cytotoxic T cells
(CTL), regulatory T cells as well as analysis of cytokines derived from asbestos-
exposed patients such as pleural plaque and mesothelioma were discussed. As well,
immunological alteration in silicosis patients who had been exposed to silica parti-
cles, induction of autoantibody production caused by mineral oil, and cell-cycle
alteration in human peripheral lymphocytes exposed by palladium nanoparticles
were presented. All the studies were very interesting and important to consider in
further proceedings in prevention and management of occupational allergy and
immunotoxicity.
The Allergy and Immunotoxicology SC has actively worked for understanding
the pathophysiology of occupational allergy and immunotoxicity, prevention, and
management of occupational environments as well as patients who have suffered
from these pathological situations. In addition, we should participate in collabora-
tive discussions, having scientific sessions with other SCs in ICOH, such as the
Respiratory Disorders SC; Rural Health: Agriculture, Pesticides, and Organic Dust
SC; Indoor Air Quality and Health SC; Occupational and Environmental Dermatoses
SC; Occupational Toxicology SC; and others. Anyone involved with these SCs,
please do not hesitate to contact our chairperson or secretary by e-mail. Let us aim
for a good session at the Dublin meeting.
Preface vii
We welcome newcomers to our SC. Anyone who is interested in allergy and

immunotoxicology issues, please contact us as soon as possible and let us support
patients with these problems. We can contribute to the future construction of better
occupational health management, especially with regard to allergy and
immunotoxicology.
Kurashiki, Japan Takemi Otsuki

Chiet, Italy Mario Di Gioacchino
Chiet, Italy Claudia Petrarca
Contents
1 Suppressive Effects of Asbestos Exposure on the Human

Immune Surveillance System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Yasumitsu Nishimura, Naoko Kumagai-Takei, Megumi Maeda,
Hidenori Matsuzaki, Suni Lee, Shoko Yamamoto, Tamayo Hatayama,
Kei Yoshitome, and Takemi Otsuki
2 Silica-Induced Immunotoxicity: Chronic and Aberrant
Activation of Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Suni Lee, Hiroaki Hayashi, Hidenori Matsuzaki,
Naoko Kumagai-Takei, Megumi Maeda, Kei Yoshitome,
Shoko Yamamoto, Tamayo Hatayama, Yasumitsu Nishimura,
and Takemi Otsuki
3 Engineered Nanomaterials and Occupational Allergy . . . . . . . . . . . . . 27
Claudia Petrarca, Luca Di Giampaolo, Paola Pedata,
Sara Cortese, and Mario Di Gioacchino
4 Allergens in Occupational Allergy: Prevention
and Management Focus on Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Mario Di Gioacchino, Luca Di Giampaolo, Veronica DAmbrosio,
Federica Martino, Sara Cortese, Alessia Gatta, Loredana Della Valle,
Anila Farinelli, Rocco Mangifesta, Francesco Cipollone, Qiao Niu,
and Claudia Petrarca
5 Particulate-Driven Type-2 Immunity and Allergic
Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Etsushi Kuroda, Burcu Temizoz, Cevayir Coban,
Koji Ozasa, and Ken J. Ishii
6 Traditional and Emerging Occupational Asthma in Japan . . . . . . . . . 83
Kunio Dobashi
ix
x Contents
7 Skin Sensitization Model Based on Only Animal

Data by Qualitative Structure-Toxicity Relationships
(QSTR) Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Kazuhiro Sato, Kohtaro Yuta, and Yukinori Kusaka
8 Nonindustrial Indoor Environments and WorkRelated Asthma . . . 103
Nicola Murgia, Ilenia Folletti, Giulia Paolocci,
Marco dellOmo, and Giacomo Muzi
9 Combined Effect on Immune and Nervous System
of Aluminum Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Qiao Niu and Qinli Zhang
10 Non Pulmonary Effects of Isocyanates . . . . . . . . . . . . . . . . . . . . . . . . . 129
Paola Pedata, Anna Rita Corvino, Monica Lamberti,
Claudia Petrarca, Luca Di Giampaolo, Nicola Sannolo,
and Mario Di Gioacchino
11 Skin Exposure to Nanoparticles and Possible
Sensitization Risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Francesca Larese Filon
Chapter 1
Suppressive Effects of Asbestos Exposure
on the Human Immune Surveillance System
Yasumitsu Nishimura, Naoko Kumagai-Takei, Megumi Maeda,

Hidenori Matsuzaki, Suni Lee, Shoko Yamamoto, Tamayo Hatayama,
Kei Yoshitome, and Takemi Otsuki
Abstract Asbestos exposure causes malignancies such as mesothelioma and lung

cancer. Asbestos induces carcinogenic activity, and its fibers may cause immune-
modifying effects that impair the immune surveillance system in regard to cancer
cell monitoring. Impairment of natural killer (NK) cells, cytotoxic T lymphocytes
(CTLs), T helper 1 (Th1) cells, and regulatory T (Treg) cells was investigated using
cell lines and freshly isolated peripheral blood immune cells derived from health
donors, as well as peripheral immune cells from asbestos-exposed patients with
pleural plaque and malignant mesothelioma (MM). All findings showed that asbes-
tos exposure caused reduction of antitumor immunity. Therefore, the carcinogenic
and immune-modifying effects indicate that the immune surveillance system in
relation to cancerous cells may be impaired by asbestos exposure.
Keywords Asbestos NK cell CTL Th1 cell Treg Immune surveillance
Y. Nishimura N. Kumagai-Takei H. Matsuzaki S. Lee S. Yamamoto T. Hatayama

K. Yoshitome T. Otsuki (*)
Department of Hygiene, Kawasaki Medical School,
577 Matsushima, Kurashiki, Okayama 701-0192, Japan
e-mail: takemi@med.kawasaki-m.ac.jp
M. Maeda
Department of Biofunctional Chemistry, Division of Bioscience, Okayama University
Graduate School of Natural Science and Technology,
1-1-1 Tsushima-Naka, Kita-Ku, Okayama 7008530, Japan
Springer Science+Business Media Singapore 2017 1

T. Otsuki et al. (eds.), Allergy and Immunotoxicology in Occupational Health,
Current Topics in Environmental Health and Preventive Medicine,
DOI 10.1007/978-981-10-0351-6_1
2 Y. Nishimura et al.
1.1 Asbestos, Malignant Mesothelioma (MM), and Immune

Function
Asbestos fibers have been used in many industrial fields worldwide because they are
a natural mineral exhibiting high flexibility; resistance against heat, fire, friction,
acids, and alkalis; as well as high electrical conductivity with a relatively low price
for supply [14]. However, the majority of advanced nations have banned the use of
asbestos due to its carcinogenicity (International Agency for Research on Cancer;
IARC categorized asbestos as a definitive carcinogen in group 1), especially its
association with lung cancer and MM, although many developing countries con-
tinue to use asbestos and several countries are currently exporting this mineral
[58].
MM is a malignant tumor occurring in mesothelial cells located in the pleura,
peritoneum, testicular serosa, and pericardium [911]. The major cause of MM is
considered exposure to asbestos, with scant cases involving exposure to uranium
and erionite (the frequent occurrence of MM in Cappadocia, Turkey, one of worlds
heritage sites, is known to be caused by erionite) [1215]. It should be noted that
MM in individuals exposed to asbestos results from relatively low to middle doses
of exposure, compared with asbestosis/asbestos-induced lung fibrosis, a type of
pneumoconiosis, which patients acquire after having been exposed to relatively
high doses (according to the asbestos fibers found in the lung, i.e., more than two
million fibers in 1 g dry lung tissue). In addition, the latent period is estimated as
3050 years from the initial exposure to asbestos [911].
The carcinogenic actions of asbestos fibers are thought to be due to (1) oxygen
stress, (2) chromosome tangling, and (3) absorption of other carcinogens in the
lung. Considering these processes, crocidolite is thought to possess the strongest
carcinogenic activity because it possesses the highest content of iron [1620]. In
addition, among the various asbestos fibers, the amphibole group, which includes
crocidolite and amosite, is considered to have a stronger carcinogenic activity than
the serpentine group, which only includes chrysotile, because of its physiological
peculiarities and rigid form, and these considerations provide a basis for the above-
mentioned carcinogenic hypotheses [1620].
However, if we consider the long latent period, there may be biological effects of
asbestos that cause malignancies other than the direct actions on alveolar and meso-
thelial cells. An insight may be gained by considering the immunological effect
because asbestos fibers are found in various lymph nodes and mainly in pulmonary
regions, not only in asbestos-handling workers, but also in individuals exposed from
the environment. In particular, investigation of individuals experiencing non-work-
related exposure revealed higher asbestos contents in lymph nodes rather than the
lung [21, 22]. The overall findings suggest a frequent association between asbestos
fibers and immune cells, and recurrent and continuous exposure to asbestos may
alter the cellular, molecular, and functional features of immune cells. A consider-
ation of malignant tumors in asbestos-exposed people indicates that the immune
effects of asbestos may comprise a reduced tumor immunity that makes individuals
more prone to cancers after a long latent period.
1 Suppressive Effects of Asbestos Exposure on the Human Immune Surveillance System 3
1.2 Alteration of Various Immune Cells Caused by Asbestos

Exposure
1.2.1 Natural Killer (NK) Cell

1.2.1.1 Human NK Cell Line, YT-A1
A human NK cell line, YT-A1, was cocultured continuously with 5 g/ml of chryso-
tile B (CB) for more than 5 months (YT-CB5). The aforementioned concentration
of CB was chosen for these experiments because it did not induce apoptosis or
growth inhibition. Meanwhile, the cytotoxicity against K562, a human erythroleu-
kemia cell line, and cell surface expression of various receptors were compared with
those of the YT-A1 original cell line, which was never exposed to asbestos. After
5 months of cultivation, YT-CB5 showed decreased cytotoxicity with reduced
expression of NK cell-activating receptors such as NKG2D and 2B4, whereas other
surface markers such as CD16, NKG2A, and CD94 were not changed. Although
killing of K562 cells is not dependent on the 2B4 receptor, YT-CB5 showed impair-
ment of 2B4-dependent cytotoxicity as analyzed by a reverse antibody-dependent
cellular cytotoxicity (ADCC) assay using the anti-2B4 antibody. In addition,
YT-CB5 showed decreased phosphorylation of extracellular signal-regulated kinase
(ERK) 1/2 after cultivation with K562 and stimulation with anti-NKG2D antibody.
These results indicate that exposure to asbestos causes a reduction of NK cell cyto-
toxicity and decreased expression in activating receptors [2328].
1.2.1.2 NK Cell Cytotoxicity in MM Patients
We investigated the state of NK cell cytotoxicity in patients with MM who might

have been exposed to asbestos through the presence or absence of an occupational
history with the mineral. After adjusting peripheral blood mononuclear cells
(PBMC) from MM patients, cytotoxicity against K562 cells caused by 5,000 NK
cells in PBMC and expression of NK cell-activating receptors were compared with
those from healthy donors (HDs). NK cells derived from MM patients showed
reduced cytotoxicity when compared with those from HD. Interestingly, although
surface expression of NKG2D and 2B4 on NK cells from MM did not alter, another
activating receptor, NKp46, exhibited reduced expression [23]. In response to this
finding, we analyzed the expression of activating receptors on NK cells derived
from HD and stimulated by interleukin (IL)-2 in vitro with or without 5 g/ml of
CB. Similar to NK cells from MM patients, there was a remarkable decrease in
NKp46 expression, whereas expression of NKG2D and 2B4 did not decrease. In
addition, the control substance used for CB, glass wool, did not induce these changes
of expression of activating receptors. Thus, asbestos exposure specifically reduced
the expression of NKp46 and resulted in reduced function of NK cells [2328].
1.2.1.3 Relationship Between Killing Activity and Receptor Expressions
The relationship between expression levels of NK cell-activating receptors with the

degree of cytotoxicity in HD and strength of downstream signaling receptors was
examined. The killing activity against K562, expression levels of activating recep-
tors, and phosphorylation levels of ERK 1/2 in NK cells derived from HD were
measured. HDs whose NK cells revealed stronger killing activity showed higher
expression of NKG2D and NKp46, as well as strong phosphorylation levels of ERK
1/2 molecules, when NK cells were simulated by anti-NKG2D or anti-NKp46 anti-
bodies. However, killing activity was not correlated with expression levels of 2B4
or phosphorylation levels of ERK 1/2 in NK cells after stimulation by anti-2B4
antibody. The overall results showed that surface expression levels of NKG2D and
NKp46 were important for cytotoxic activity in NK cells derived from asbestos-
exposed patients. The overall results showed that the impairment of NK cells in
these patients may be caused by decreased expression of these molecules induced
by the continuous and recurrent exposure to asbestos [2328].
1.2.2 Cytotoxic T Lymphocyte (CTL)
1.2.2.1 CTL Differentiation and Proliferation in Mixed Lymphocyte

Reaction (MLR)
The MLR assay was used to evaluate CTL function derived from nave CD8+ T
cells cultured with allo-PBMC or splenic cells in vitro. Thus, to examine the effects
of CB on CTL function, PBMC derived from HD were utilized for MLR using allo-
PBMC with or without 5 g/ml of CB. The increase of CD8+ cells in MLR was
suppressed by addition of CB and cytotoxicity targeting the allo-PBMC using sorted
CD8+ cells after cultivation was also reduced. Consistent with these findings, intra-
cellular positivity of effector molecules such as granzyme B and interferon (IFN)-
in CD8+ cells cultured with CB during the MLR was reduced. Regarding the dif-
ferentiation of CD8+ cells, CD45RA as the marker for nave CD8+ cells remained
relatively high, while CD25 and CD45RO as markers of effector/memory CD8+
cells were not elevated when cultured with CB, compared with no CB MLR. In
addition, the proliferation of CD8+ cells examined by the carboxyfluorescein suc-
cinimidyl ester (CFSE) labeling method was also inhibited when cultured with CB,
although apoptotic cells of CD8+ cells were not changed regardless of whether the
MLR was cocultured with or without CB. Furthermore, production of tumor necro-
sis factor (TNF)- and IFN- in culture supernatants decreased when MLR was
performed with CB, whereas IL-2 did not change irrespective of the presence of
CB. These findings show that asbestos exposure causes inhibitory effects on CTL
induction from CD8+ nave T cells [2931].
1.2.2.2 Characteristics of CTL in Asbestos-Exposed Patients
CTL differentiation and proliferation were impaired by asbestos exposure in

asbestos-exposed patients with MM and pleural plaque (PP), who showed plaque in
pleura as the marker of asbestos exposure and no other health effects in their body.
The status of CTL characteristics in PP and MM patients was not identical. Analysis
of CD8+ cells from PP patients revealed an increase of the perforin-positive and
CD45RA-negative population in peripheral blood, as well as an increase of the per-
forin- and IFN--positive fraction after in vitro stimulation, whereas MM patients
showed a decrease of perforin-positive CD8+ cells in peripheral blood. Furthermore,
this reduction was not due to the excess degranulation.
Details regarding the features of CTL in PP and MM were published in our pre-
vious reports. In particular, the reduction of killing activity in CD8+ cells caused by
asbestos exposure suggests that the pathophysiological status such as the presence
or absence of cancers may alter the function of CTL. This issue needs to be resolved
in future studies [2931].
1.2.3 Alteration of T Cell Caused by Asbestos Exposure
1.2.3.1 T Helper 1 (Th1) Cell
Asbestos Exposure on Human T Cell Line, MT-2
A human T cell line, MT-2 (human T cell leukemia/lymphoma virus 1 (HTLV-1)

immortalized polyclonal T cell line), was utilized to establish a continuous and
recurrent asbestos exposure model (more than 1 year exposure to low-dose asbestos
fibers and details were reported previously [32]). Six independent continuously
exposed sublines of MT-2 cocultured with CB were established, and cDNA micro-
array analysis was performed to compare the sublines with the original MT-2 cells,
which were never exposed to asbestos. All six sublines showed a similar expression
pattern of cDNA, and 84 up- and 55 downregulated genes were specified. Network
analysis using the MetaCore system indicated that the IFN- pathway was involved
in the sublines. All sublines showed a decrease in IFN regulatory factor 9 (IRF9)
and IFN-stimulating gene factor 3 (ISGF3), as well as a decrease of CXC chemo-
kine receptor 3 (CXCR3), which was regulated by IRF9. Since it is known that the
Th1 cell shows higher IFN- and CXCR3 expression, mRNA and protein expres-
sion in the MT-2 sublines were reanalyzed. Results indicated that all sublines
showed a decrease of CXCR3 and IFN- productive capacity, whereas the other
Th1-type chemokine receptor, C-C chemokine receptor type 5 (CCR5) which had
been chosen as the comparison, did not change [3335].
CXCR3 and IFN- in Peripheral Th1 Cells Cultured with CB
Following analysis of the cell line model, in which it was supposed that asbestos
exposure may inhibit Th1 function, and to confirm these findings in freshly isolated
CD4+ Th cells from HD, these cells were stimulated and activated using anti-CD3
and anti-CD28 antibodies with IL-2 and cocultured with or without 5 or 10 g/ml of
CB. The CB-exposed Th cells showed decreased surface and mRNA expression of
CXCR3 and intracellular IFN--positive cells after 4 weeks of cultivation. These
results indicated that ex vivo exposure to asbestos causes a distinct reduction of Th1
function as revealed by expression of CXCR3 and IFN- [3335].
CXCR3 Expression of Peripheral CD4+ Cells in MM and PP Patients
In response to the experimental results of the cell culture, the expressions of CXCR3
and IFN- were analyzed in peripheral blood CD4+ cells derived from PP and MM
patients, and results were compared with those from HD. Results showed that the
CXCR3 expression level in CD4+ cells from PP and MM patients decreased and
that of MM was lower than that of PP, whereas expression of CD4+CCR5+ cells in
peripheral blood did not differ between the PP, MM, and HD groups. Moreover,
CD4+ Th cells derived from MM showed decreased mRNA expression of IFN-
when cells were ex vivo stimulated. A consideration of the overall results and exper-
imental findings indicates that clinical exposure to asbestos induces dysfunction of
Th1 cells as specifically revealed by decreased expression of CXCR3 and IFN-.
Since both CXCR3 and IFN- are known to be important for antitumor immunity,
these findings support the hypothesis that asbestos exposure induces a gradual
decrease of antitumor immunity in the body of an exposed individual, which even-
tually causes lung cancer and MM in these individuals after a long latent period
following the initial exposure to asbestos [3335].
1.2.3.2 Regulatory T Cell (Treg)
In Vitro Assessment of Treg Function Exposed to Asbestos Using MT-2
The MT-2 cell line is known to possess Treg functionality, since HTLV-1 has a high
affinity for Treg cells and adult T cell leukemia/lymphoma is considered a malig-
nancy of Treg [3638]. Treg is important for various pathophysiological states; for
example, if the quality or quantity of Treg is decreased, allergy and autoimmune
disorders may occur because the reaction of responder T cells against self- or non-
self-antigens is not suppressed by Treg. In addition, if the function or volume of
Treg is upregulated, antitumor immunity may decrease because responder T cells
that recognize the tumor antigen and CTL suppress their function [3941].
The abovementioned cell line model continuously exposed to asbestos was

established using MT-2, and analyses of Treg function using the MT-2 sublines and
the original MT-2 cell line, which was never exposed to asbestos fibers, may clarify
the alteration of Treg following asbestos exposure [42].
Treg expresses suppressive activity by cell-cell contact and soluble factors, typi-
cally known as IL-10 and transforming growth factor (TGF). Interestingly, all sub-
lines showed overproduction of IL-10 and TGF. The overproduced IL-10 was
regulated by Src family kinases in MT-2 because of the reduction of IL-10 produc-
tion and expression by the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-
butyl)pyrazolo[3,4-d]pyrimidine (PP2). This IL-10 was utilized by MT-2 cells
through autocrine mechanisms and caused phosphorylated activation of signal
transducer and activator of transcription (STAT)3, as well as Bcl-2 located down-
stream of STAT3. These conditions made the sublines resistant to asbestos-induced
apoptosis. In addition, the overproduced TGF resulted in TGF-induced growth
inhibition in MT-2 original cells, with over-phosphorylation of the downstream sig-
naling molecule p38, as well as the TGF pathway signaling molecule SMAD3, but
resulted in a decrease of SMAD2 [32, 43].
The contact inhibitory function of the MT-2 sublines exposed continuously to
asbestos was then examined. Freshly isolated CD4+ cells were stimulated with anti-
CD3 and auto-peripheral blood monocyte-derived dendritic cells to induce prolif-
eration of cells. Instead of autologous Treg, irradiated cells from the original MT-2
cell line or an asbestos-exposed subline were added to this culture. Results revealed
that the subline showed a greater enhanced suppressive activity than the original
MT-2 cells, indicating that asbestos exposure caused an increase of Treg function
[42].
Furthermore, the overproduced soluble factors IL-10 and TGF were evaluated
by individually knocking down both cytokines using the siRNA method. The above-
mentioned assay for Treg suppressive function was then performed using a tran-
swell culture plate with the MT-2 subline, MT-2 original cells, and IL-10 or TGF
knocked-down sublines and permitting only the soluble factor to affect CD4+ cell
proliferation activity. Results indicated that the IL-10 and TGF knocked-down
sublines had reduced suppressive activity. These findings show that asbestos-
exposed Treg exhibits an enhanced inhibitory function by a cell-cell contact mecha-
nism, as well as excess production of functional soluble factors [42].
Analysis of the number and/or function of Treg may be better for an evaluation
of tumor-surrounding Treg, although we have not had the opportunity to analyze
Treg surrounding MM or asbestos-induced lung cancer cells. Our current findings
are therefore limited due to the experimental assay. However, asbestos may cause
the reduction of antitumor immunity by altering Treg function. The examination of
the level of proliferating activity and inhibitory function of Treg should be per-
formed in future investigations [42].
1.3 Summary of Asbestos-Induced Reduction of Antitumor

Immunity
Table 1.1 summarizes investigation of the experimental immunological effects of

asbestos exposure on various immune cells and alteration of immune function in
asbestos-exposed patients with PP and MM. Additionally, Fig. 1.1 summarizes the
typical findings described in this chapter.
As mentioned at the beginning of this chapter, the carcinogenic actions of asbes-
tos fibers are attributed to (1) oxygen stress, (2) chromosome tangling, and (3)
absorption of other carcinogens in the lung [1620]. Due to these or other mecha-
nisms, mesothelial cells may tend to change their cellular and molecular character-
istics toward an abnormal and transformed cell type. For example, p16
cyclin-dependent kinase inhibitor, NF2, neurofibromatosis type 2, BAP1, and breast
cancer susceptibility gene 1 (BRCA1 )-associated protein-1 (ubiquitin carboxy-
terminal hydrolase) are the typical altered tumor suppressor genes in MM [4448].
However, many of these transforming cells are usually monitored by immune sur-
veillance and then removed from the body. However, asbestos-exposed individuals
may possess an impaired immune surveillance system as described in this chapter,
Table 1.1 Investigations of the experimental immunological effects of asbestos exposure on

immune cells and alteration of immune function in asbestos-exposed patients
Kinds of asbestos
fiber for exposure or
analyzed patients
Immune cell type exposed to asbestos Findings References
NK cell [2328]
Human NK cell Cultivation with Reduction of cytotoxicity
line, YT-A1 chrysotile Reduction of surface expression of
NKG2D and 2B4
Decreased phosphorylation of ERK
signaling molecule
Peripheral CD56+ Malignant Low cytotoxicity
NK cells mesothelioma Low surface expression of
NKp46-activating receptor
Freshly isolated Cultivation with Reduction of surface expression
NK cells derived chrysotile during of NKp46
from healthy in vitro activation
donors
Cytotoxic T cell [2931]
Human CD8+ Cultivation with Reduction of allogenic cell killing
cells in mixed chrysotile during Decrease of intracellular IFN- and
lymphocyte MLR granzyme B
reaction (MLR)
Peripheral CD8+ Pleural plaque Relatively high perforin+ cell
T cell
(continued)
Table 1.1 (continued)

Kinds of asbestos
fiber for exposure or
analyzed patients
Immune cell type exposed to asbestos Findings References
T helper cell [3234, 42,
Human T cell line, Continuous Acquisition of asbestos-induced 43]
MT-2 cultivation with apoptosis
chrysotile Excess expression and production
of IL-10
Overexpression of Bcl-2
Reduced production of IFN-,
TNF-, and CXCL10
Reduction of CXCR3 surface and
mRNA expressions
Hyperphosphorylation of -actin
Excess binding capacity to
chrysotile in vimentin, myosin 9,
and tubulin2
Excess production of TGF with
phosphorylation of p38 and
SMAD3
Resistance to TGF-induced
growth inhibition
Continuous Acquisition of asbestos-induced
cultivation with apoptosis
crocidolite Excess expression and production
of IL-10
Enhancement of Bcl-2/Bax
expression ratio
Reduced production of IFN- and
TNF-
Freshly isolated Cultivation with Reduced expression of surface
CD4+ cells chrysotile during CXCR3
derived from in vitro activation Reduction of intracellular IFN-
healthy donors
Peripheral CD4+ Pleural plaque Low expression of surface
T cell CXCR3
Malignant Remarkably lower expression of
mesothelioma surface CXCR3
Low IFN- mRNA expression
High Bcl-2 mRNA expression
Regulatory T cell [41]
Human T cell line, Continuous Enhanced suppressive activity in
MT-2 cultivation with cell-cell contact assay
chrysotile Enhanced production of
functional soluble factors such as
IL-10 and TGF
Fig. 1.1 Summarized schematic effects of asbestos fibers on various immune cells such as natural
killer (NK), cytotoxic T lymphocyte (CTL), nave CD8+, T helper 1 (Th1), and regulatory T (Treg)
cells (right side of figure). The carcinogenic effects of asbestos fibers are shown on the left side,
and normal mesothelial cells are gradually transformed toward malignant mesothelioma cells with
alteration of tumor suppressor genes such as p16, NF2, and BAP1. Between these two effects, the
usual immune surveillance system regarding cancerous cells may be impaired by asbestos
exposure
and this impairment may result in MM and other cancers in these individuals after
a long latent period [4953].
Future investigations aimed at neutralizing the immune surveillance system in
the asbestos-exposed population through physiologically active substances in foods,
plants, and other materials are necessary in order to prevent the occurrence of can-
cerous diseases in asbestos-exposed individuals.
Acknowledgments The authors thank Ms. Minako Katoh, Naomi Miyahara, Satomi Hatada,
Keiko Yamashita, Keiko Kimura, Tomoko Sueishi, and Misao Kuroki for their technical assis-
tance. All the experimental findings performed in the Department of Hygiene, Kawasaki Medical
School, were supported by the Special Coordination Fund for Promoting Science and Technology
grant H18-1-3-3-1; JSPS KAKENHI grants 17790375, 19790431, 20890270, 22790550,
23790679, 24590770, and 25860470; Kawasaki Medical School Project grants 29-403, 19-407 M,
20-402O, 20411I, 32-107, 21-401, 22A29, 22B1, 23P3, 23B66, 24B39, and 25B41; the Kawasaki
Foundation for Medical Science and Medical Welfare (2007 and 2009); and the Ryobi Teien
Memorial Foundation (2009 and 2010).
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Chapter 2
Silica-Induced Immunotoxicity: Chronic
and Aberrant Activation of Immune Cells
Suni Lee, Hiroaki Hayashi, Hidenori Matsuzaki, Naoko Kumagai-Takei,

Megumi Maeda, Kei Yoshitome, Shoko Yamamoto, Tamayo Hatayama,
Yasumitsu Nishimura, and Takemi Otsuki
Abstract Occupational and chronic exposure to silica particles causes pulmonary

fibrosis known as silicosis, which is a typical form of pneumoconiosis. Although the
respiratory complications of silicosis include pulmonary tuberculosis, pleurisy,
bronchitis, and other disorders such as lung cancer, silicosis (SIL) patients often
suffer from complications of autoimmune diseases such as rheumatoid arthritis
(RA), systemic sclerosis (SSc), and other forms of these conditions. The mecha-
nisms causing impairment of immunity by silica exposure were considered adjuvant
effects of these particles, and we have been investigating the direct effects of parti-
cles on immune cells, particularly T cells, and found chronic activation of both
responder and regulatory T helper cells. The results of chronic activation indicated
that responder T cells obtain resistance against CD95/Fas-mediated apoptosis to
survive longer, whereas regulatory T cells become prone to CD95/Fas-mediated
apoptosis and die quickly. These findings suggest that the imbalance of these cells
may initiate the observed autoimmune disorders. Furthermore, various autoantibod-
ies were detected in the serum of SIL without any symptoms of autoimmune dis-
eases. Further investigation is needed to study the effects of silica on other immune
cell types such as Th17, dendritic, and B cells. Adequate preventive tools against the
progression of immunological impairments are also required to improve occupa-
tional health.
S. Lee H. Matsuzaki N. Kumagai-Takei K. Yoshitome S. Yamamoto T. Hatayama

Y. Nishimura T. Otsuki (*)
Department of Hygiene, Kawasaki Medical School,
e-mail: takemi@med.kawasaki-m.ac.jp
H. Hayashi
Department of Dermatology, Kawasaki Medical School,
M. Maeda
Department of Biofunctional Chemistry, Division of Bioscience, Okayama University
Graduate School of Natural Science and Technology,
1-1-1 Tsushima-Naka, Kita-Ku, Okayama 7008530, Japan

DOI 10.1007/978-981-10-0351-6_2
16 S. Lee et al.
Keywords Silica Autoimmune diseases Regulatory T cell CD95/Fas

Apoptosis
2.1 Silica Exposure and Pulmonary Diseases
Silica-exposed patients suffer from pulmonary fibrosis known as silicosis, a typical

form of pneumoconiosis [15]. Although silicosis is divided clinically into chronic,
subacute, and acute forms, simple silicosis is characterized by the presence of small
rounded shadows, which usually appear in the upper lobes and later are observed
throughout the lungs [15]. This is a classic disease historically and was noted in the
era of Hippocrates, as well as Ramazzini and Agricola in their books De Morbis
Artificum Diatriba and De Re Metallica [13]. They wrote that miners developed
dyspnea and suggested a positive relationship between rock-dust exposure and dys-
pnea. The occupational sources of exposure are mining, quarrying (granite, sand-
stone, slate, pumice, pumicite), tunneling, stone working, activities involving
abrasives, glass manufacture, the agate industry and glass manufacture, the refrac-
tory brick industry, and many other sources [16].
The pulmonary complications of silicosis are typically tuberculosis, as well as
pleuritis, pneumothorax, bronchitis, bronchiectasis, and lung cancer. Interestingly,
the International Agency for Research on Cancer (IARC) of the World Health
Organization (WHO) categorized crystalline silica as a group I carcinogen when
inhaled in the form of quartz and cristobalite from occupational sources [710].
Basically, there are no curative procedures for chronic progression of lung fibro-
sis designated as silicosis, and these patients suffer from dyspnea and other pulmo-
nary symptoms [15].
2.2 Immune Complications
In addition to the pulmonary complications mentioned above, SIL suffer from auto-
immune disorders [13, 1115]. Caplans syndrome [16, 17] and rheumatoid arthri-
tis (RA) are well-known complications of silicosis. Systemic sclerosis (SSc) [1821]
and systemic lupus erythematosus (SLE) [2224] are also known as frequent com-
plications of silicosis, although SLE appears to be limited to workers associated
with sandblasting, grinding, and the handling of silica for use as a scouring powder
or in patients with acute or accelerated silicosis [13]. More recently, anti-neutrophil
cytoplasmic antibody (ANCA)-related vasculitis/nephritis have been reported as
complications in SIL [2528].
Basically, the cellular mechanisms responsible for the dysregulation of autoim-
munity caused by silica exposure have been regarded as an adjuvant effect of the
silica particles [2931]. There are many self-antigens such as proteins, amino acids,
2 Silica-Induced Immunotoxicity: Chronic and Aberrant Activation of Immune Cells 17
and nucleotides derived from cell debris yielded by physiological cell apoptosis and
other processes in the body. These candidate antigens may bind with the silica par-
ticles as adjuvants and subsequently exert strong antigenicity to induce the autoim-
mune diseases observed in SIL.
However, since inhaled silica particles remain in the lung and lymph nodes, these
particles may recurrently and chronically encounter circulating immune cells [15].
It is therefore reasonable to consider the possible direct effects of these inhaled sil-
ica particles on the peripheral immune cells of the human body.
2.3 Alteration of Immune Cells and Functions
2.3.1 Responder T Cell Activation Caused by Silica
Although it is commonly thought that immunotoxicity may kill immune-competent

cells through the action of certain substances, our investigations have revealed
excess immune stimulation or reduction of certain immune levels due to environ-
mental factors such as silica particles to result in representative phenotypes of
immunotoxicity when considering that immune systems should remain physiologi-
cally neutral. Excess immune reactions cause allergies and autoimmune disorders,
and a reduced immunological state may result in impairment of tumor immunity
and transplantation immunity. Thus, a consideration of immune impairments caused
by environmental substances suggests that altered activation and/or reduced func-
tion of immune cells should be regarded as an indication of immunotoxicity in the
broad sense of the term.
In vitro experiments were performed initially to determine whether silica parti-
cles produced immunotoxic effects on human peripheral T cells [32]. When periph-
eral blood mononuclear cells (PBMCs) were cultured with or without silica particles,
only T cells in PBMC gradually expressed CD69 as an early activation marker of T
cells for 410 days, although the increased expression of CD69 was slower than that
of standard mitogen such as phytohemagglutinin (PHA), which immediately acti-
vated T cells within 24 h followed by cessation within 2 or 3 days. Actually, chryso-
tile asbestos fibers did not allow T cells to express CD69 [32].
In addition, PD-1 expression as one of the chronically activated T cell markers
was examined using peripheral blood T cells (CD4+ and CD25+ or CD4+ CD25-
fractions) from SIL and compared with that of healthy volunteers (HV). Both frac-
tions derived from SIL showed a significantly higher expression of PD-1 than that
of HV [33].
Since the concentration of serum soluble interleukin (IL)-2 receptor (sIL-2R) has
recently been considered an activation marker of T cells in autoimmune diseases, in
addition to tumor biomarker for T cell leukemia and lymphoma [34, 35], serum sIL-
2R levels in HV, SIL, and SSc were measured and compared [36]. Results revealed
that sIL-2R levels in SSc were significantly higher than those in HV, and levels in
18 S. Lee et al.
SIL tended to be higher than those in HV. In addition, the level of sIL-2R in SIL was
correlated with antinuclear antibody (ANA) titer, immunoglobulin (Ig) G, and anti-
centromere/CENP-B antibody titer. Furthermore, factor analysis indicated that sIL-
2R in SIL contributed to the subpopulation with a poorer immunological status
without much impairment of respiratory parameters [36]. Thus, sIL-2R was also
considered an indicator of the chronically activated status of T cells in SIL.
The overall findings suggested that responder T cells in SIL are chronically acti-
vated by recurrent encounters with silica particles in the circulation.
2.3.2 Inhibitory Molecules for CD95/Fas-Mediated Apoptosis

in SIL
As mentioned above, responder T cells circulating in the peripheral blood of SIL are
activated chronically. However, the reactions of activated T cells usually cease due
to activation-induced cell death (AICD) mediated by CD95/Fas death receptor via
autocrine or paracrine binding with Fas ligand produced by similarly activated T
cells [3739]. Examination of autoimmune diseases revealed that HV had lower
serum levels of soluble Fas (sFas), which lacks the transmembrane domain of the
Fas molecule as an alternatively spliced variant and is secreted from cells for bind-
ing with the auto- or para-produced Fas ligand at extracellular spaces to prevent
Fas-mediated apoptosis/AICD [4043]. If the chronically activated T cells in these
diseases, which are recognizing the self-antigens, are avoiding AICD/Fas-mediated
apoptosis, these self-recognizing T cells may survive longer and cause continuous
impairment of autoimmunity to result in autoimmune disorders that last longer and
progress to worsening states.
Serum sFas levels in SIL were also elevated, and examination of mRNA expres-
sion in PBMC revealed excess expression of the sFas message, which lacks the 63
bp of the transmembrane domain relative to the wild-type membrane and binds the
Fas molecule [44, 45]. In addition, PBMC from SIL showed a higher expression
relative to HV of other alternatively spliced variants of the Fas gene, all of which
conserved the Fas-ligand binding domain but lacked the transmembrane domain
[46]. Most of these variants may act in a manner similar to the typical variant, the
sFas molecule, to prevent Fas-mediated apoptosis.
Similar to sFas, decoy receptor 3 (DcR3) was also found to prevent TNF-related
apoptosis-inducing ligand (Trail)-mediated apoptosis by binding with Trail at extra-
cellular spaces [4749]. DcR3 molecules were first reported to be produced by lung
and colon cancer cells for self-prevention from tumor attacking Trail secreted by T
cells bearing tumor immunity [50]. Thereafter, serum DcR3 levels were found to be
higher in various autoimmune diseases. In our analysis, PBMC from SIL showed a
higher DcR3 expression level than that of HV [51]. We have been trying recently to
measure serum DcR3 levels, as well as determine the role of DcR3 levels in the
immunological pathophysiological status of SIL. We will report the findings of

these investigations in the near future.
The overall results indicate that responder T cells chronically activated by silica
particles avoid AICD/Fas-mediated apoptosis by self-producing inhibitory
molecules.
2.3.3 Regulatory T Cells in Silicosis Peripheral Blood
When we consider the T cell status that tends to progress to autoimmune disorders,
the balance between responder T cells and regulatory T cells (Treg) defined by
CD4+ CD25+ and forkhead box P3 (FoxP3) transcription factor positive is impor-
tant, and reduction of function and/or number of Treg may prolong the activation of
responder T cells from various non-self or self-antigens and cause autoimmune dis-
orders [52, 53]. In addition, T helper 17 (Th17) cells are also important in the for-
mation of autoimmune disorders [5456]. Cytokine levels, particularly those of
IL-6 and transforming growth factor (TGF-)-, influence the polarization between
Treg and Th17 cells [5759]. Although we have not investigated Th17 alteration
following exposure to silica particles, we found an interesting change in Treg fol-
lowing exposure to silica particles, as well as alteration of peripheral Treg from SIL
[60].
We first examined Treg function derived from the CD4+CD25+ fraction of SIL
and compared it with that of HV [60]. Although the examined SIL did not show any
symptoms of autoimmune diseases, the suppressive function that reduces the
antigen-induced proliferation in responder (CD4+CD25-) T cells induced by a
cocultured peripheral blood CD4+CD25+ fraction was reduced in SIL compared to
HV. This result indicated that Treg function in the peripheral CD4+CD25+ fraction
of SIL may be reduced or weakened by chronic exposure to silica particles [60].
Treg from SIL and HV showed higher CD95/Fas expression compared with that
in responder T cells of the CD4+CD25- fraction [33]. However, it was significantly
higher in SIL compared to HV. Since CD95/Fas expression in Treg is known as an
activation marker for this cell type, chronic silica exposure may stimulate Treg in a
manner similar to that of responder T cells as mentioned above. The results showing
chronic activation of Treg with excess expression of CD95/Fas may indicate they
are sensitive to Fas-mediated apoptosis [33]. In fact, Treg from SIL proceeded
toward apoptosis more rapidly and with greater amounts compared to Treg from HV
when these cells were cultured with agonistic anti-Fas antibody. Furthermore, cul-
tures with silica particles and PBMCs from HV revealed that the CD4+CD25+ frac-
tion as a marker of Treg and activated responder T cells did not change remarkably;
however, the level of FoxP3+ in CD4+ decreased significantly during the culture
period [33]. These results indicate that Treg in SIL are highly sensitive to
Fas-mediated apoptosis and can be killed easily and may be recruited from the bone
marrow [33].
20 S. Lee et al.
The overall findings show that silica exposure chronically activates responder T
cells and Treg. The former are resistant to Fas-mediated apoptosis and survive lon-
ger, whereas the latter are sensitive and progress toward cell death. An imbalance
between these two types of T cells then occurs in SIL and subsequently results in the
germination of impaired autoimmunity.
2.3.4 Autoantibodies in SIL
We have reported various autoantibodies that were found in SIL. For example, anti-
CD95/Fas autoantibody was found in approximately one fourth of SIL [61]. This
autoantibody was functional, i.e., when this autoantibody binds with membrane
Fas, the cells proceed toward apoptosis. This was confirmed using sister human
myeloma cell lines from pleural effusion and the bone marrow derived from the
same patients [61, 62]. The cell line from the effusion showed scant Fas expression,
whereas the line from the bone marrow exhibited strong expression. Additionally,
the former line was not killed by serum from anti-Fas autoantibody positive SIL, but
cells in the latter line died. These results indicated that Treg in SIL who are positive
for this autoantibody are much more sensitive and likely to be killed.
We have also reported anti-caspase 8 [63, 64] and anti-desmoglein autoantibod-
ies [65] in SIL. Moreover, anti-Scl-70 (topoisomerase I) autoantibody was detected
in SIL [66, 67]. These autoantibodies are clinically significant and are often detected
in SSc. The anti-Scl-70 autoantibody is seen in the diffuse SSc type with organ
fibrosis such as the lung, whereas the anti-CENP-B (centromere) autoantibody is
observed in the limited type of SSc with lesions in the esophagus and involving
pulmonary hypertension. The clinical status of both antibodies was assayed in SIL
patients that did not exhibit any symptoms of SSc. Recently, the titer index (Log10)
of anti-CENP-B autoantibody in SIL was higher than that of HV, and that of SSc
was higher than those of HV and SIL. This titer index was positively correlated with
an assumed immune status of one for HV, two for SIL, and three for SSc. Moreover,
factor analysis of SIL cases revealed that although the titer index of anti-CENP-B
autoantibody formed the same factor with that of anti-Scl-70 autoantibody, the Ig G
value and the age of patients and other factors extracted showed that the Ig A value
and anti-Scl-70 antibody were positively related, but anti-CENP-B showed an oppo-
site tendency [68]. These results indicated that the titer index of anti-CENP-B auto-
antibody may be a biomarker for dysregulation in SIL cases.
Our findings suggest that B cells, as the producer of autoantibodies, may be
affected by chronic silica exposure and an alteration caused by long-surviving
responder T cells. In addition, dendritic cells as the initial antigen-presenting cell
may have their function modified by chronic exposure to silica, although we have
not performed a detailed analysis of the cellular and molecular alterations of these
cells following silica exposure.
2.4 Conclusion
All the findings described above are summarized schematically in Fig. 2.1. Silica
exposure causes a form of pulmonary fibrosis known as silicosis, a condition that
progresses gradually to reduce the health of affected individuals. In addition, the
pulmonary complications observed with this condition sometimes result in a severe
pathological status, particularly when involving tuberculosis and lung cancer, which
further burdens SIL patients with a difficult clinical course. However, silica
Fig. 2.1 Schematic presentation of the immunological effects of silica particles, particularly on
responder T cells and regulatory T cells, and the detection of various autoantibodies
22 S. Lee et al.
particles not only cause respiratory impairments, but also immunological disorders,
especially those involving autoimmune diseases [6874]. Based on the adjuvant
effect of silica particles, our investigations have elucidated the direct action of silica
on immune-competent cells. Silica chronically activates responder and regulatory T
cells to result in an imbalance of these two types of T cells, which makes individuals
more prone to developing autoimmune disorders. Once autoimmune impairments
appear, the pathological status also progresses and worsens gradually, never to
return to the previous unimpaired condition. Although future studies are required
regarding silicas direct effects on Th17, dendritic, and B cells, investigations of
preventive procedures using physiologically active substances or chemicals from
plants and foods are necessary to inhibit the subclinical progression of immunologi-
cal impairments and improve occupational health.
Acknowledgments The authors thank former colleagues in the Department of Hygiene, Kawasaki
Medical School, namely, Prof. Ayako Ueki, Drs. Fuminori Hyodoh, Akiko Takata-Tomokuni,
Yasuhiko Kawakami, Takaaki Aikoh, Shuko Murakami, and Yoshie Miura. We also appreciate the
technical assistance of Ms. Haruko Sakaguchi, Naomi Miyahara, Minako Katoh, and Yumika
Isozaki. We express special thanks to Drs. Masayasu Kusaka and Kozo Urakami for coordinating
the collection of clinical samples. Part of the experimental results in this article was supported by
the Special Coordination Fund for Promoting Science and Technology (H18-1-3-3-1,
Comprehensive approach on asbestos-related diseases), KAKENHI grants (18390186,
19659153, 20390178, and 25460825), Kawasaki Medical School Project grants (20-410I, 23S5,
24S6, 25B65, and 27B06), the Sumitomo Foundation Grant (053027), the Yasuda Memorial
Foundation Grant (H18), funding from the Takeda Science Foundation (I-2008) and Young
Investigator Activating Grant from the Japanese Society of Hygiene (H18), the Ryobi Teien
Memorial Foundation (H24), and the Kawasaki Foundation for Medical Science and Medical
Welfare (H24).
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Chapter 3
Engineered Nanomaterials and Occupational
Allergy
Claudia Petrarca, Luca Di Giampaolo, Paola Pedata, Sara Cortese,

and Mario Di Gioacchino
Abstract The expanding demand for highly performing devices and products has
prompted the development of innovative nanosized materials. This fact, along with
the uncertain efficacy of protective equipment available for the nanosized particles,
poses concern about the risk of becoming sensitised in the occupational setting.
Actually, such a phenomenon can also affect the general population given the
unavoidable environmental contamination. At the same time, studies on the physio-
pathology of the allergy demonstrated that the lack of prevention and treatment can
result in invalidating diseases that, in case of professional aetiology, might imply
removal from job and compensation. The potential role of nanomaterials in the
development and exacerbation of occupational allergy is being disclosed by recent
C. Petrarca (*)
Immuntotoxicology and Allergy Unit & Occupational Biorepository,
Center of Excellence on Aging and Translational Medicine (CeSI-MeT), G. DAnnunzio
University Foundation, Chieti, Italy
e-mail: c.petrarca@unich.it
L. Di Giampaolo
Department of Medical Oral and Biotechnological Science, G. dAnnunzio University,
Chieti, Italy
P. Pedata
Department of Experimental Medicine, Section of Hygiene, Occupational Medicine and
Forensic Medicine, Second University of Naples, Naples, Italy
S. Cortese
Department of Medicine and Science of Aging, G. dAnnunzio University, Chieti, Italy
M. Di Gioacchino

DOI 10.1007/978-981-10-0351-6_3
28 C. Petrarca et al.
experimental investigations in cellular and animal models. Moreover, first emerging

data from professional human exposure are adding new information to the complex
puzzled picture of nanotoxicology.
Most importantly, a deeper knowledge on the role of nanomaterials in the aetiol-
ogy of the allergic diseases will allow the implementation of risk assessment and
preventive measures for nanosafety at the workplace. Original articles retrieved from
PubMed and Google searches using the keywords, occupational, exposure, workers,
nanoparticles, nanomaterials, allergy as well as congress proceedings, institutional
reports and unpublished data from research laboratories involved in this field have
been considered as a source of updated information to write this chapter.
Keywords Nanomaterials Nanoparticles Allergy Occupational Workers

Nanosafety Health surveillance Research
3.1 Introduction
Nowadays, allergic diseases affect more than one third of the word population.
Amongst them, those with an occupational cause appear to be common and are
increasing, as observed by a recent esteem of the worldwide prevalence ranging
from 5 to 15 % [1, 2]. Asthma, rhinitis, conjunctivitis, urticaria and contact derma-
titis can be allergic, triggered by occupational factors such as high and low molecu-
lar weight substances acting as complete antigens or haptens. Moreover, according
to epidemiological and experimental evidences, respiratory allergy could induce an
inflammatory process leading to pulmonary function decline, which is a highly
threatening condition exacerbated by the persistence of occupational allergen expo-
sure [3]. Engineered nanomaterials, intentionally designed with specific properties
for a wide range of technological applications, might contribute to the increasing
prevalence of those diseases amongst workers [4]. In fact, due to the rapid advance-
ments in this field, workers from nanotech industries and research laboratories
might be exposed to these new materials. In this regard, occupational health risks
associated with nanomaterials are still undefined, and little information is available
on main exposure routes, potential exposure levels and toxicity [5]. The profes-
sional exposure is likely to occur during the post-production phase, while the reac-
tion chamber is opened, when the product is handled, or during the cleanout
procedures [6]. At research workplaces, the handling of dry powders of nanoparti-
cles can generate airborne nanoparticles able to reach the breathing zone [7]. A
recent Dutch study addresses many different categories of workers being potentially
at risk of exposure to nanomaterials in that country [8].
So far, it has been difficult to assess the level of environmental contamination
and the internal dose in exposed workers due to recent introduction of measure
devices, still under evaluation, and lack of bioassays. It is well established, for
instance, that aggregates and agglomerates, rather than solitaire nanoparticles, are
the main components of a nano-substance within a fluid mean [9], which result
3 Engineered Nanomaterials and Occupational Allergy 29
difficult to split apart because of the shape and electrostatic charge. Therefore, these
multi-nanoparticle entities are difficult to assess by the current measurement
devices, nor it is possible to establish to which extent they can break up into smaller
units in the lung fluid [10]. Generally, as we also do in this chapter, the term
nanoparticles (NPs) is used to describe all the forms.
The assessment of the exposure level to NPs is made even more difficult for the
copresence of other particles of different types, giving a background signal and the
possible temporal changes in concentration [6].
An even more complex scenario comes from the fact that airborne chemicals can
be adsorbed onto (nano)particles and gain new bioactive functions, as for the immu-
nomodulatory effects characterising PM 0.5 (which include NPs) in industrial and
traffic-influenced urban areas [11].
Nevertheless, some data are emerging on NP detection in biological matrixes of
exposed workers. For instance, airborne titanium dioxide NPs were found in 40 %
of pre-shift samples and 70 % of the post-shift samples of exhaled breath conden-
sate of production workers, indicating the persistence of NPs; moreover, 10 % of the
post-shift urine samples contained NPs, confirming their translocation through the
body [12]. It is well established that NPs can enter the human body upon exposure
through inhalation, ingestion and absorption through the skin and reach tissues and
cells [13]. Then, NPs can be included within exosomes, a sort of Trojan horse that
allows them to spread from the capturing cells (macrophages and epithelial or endo-
thelial cells) into the bloodstream and other cell types and tissues [14]; for metal-
based NPs, there can be release of ions [15]. The case of cobalt is emblematic since
NPs made of this metal rapidly release ions (Co2+) in culture medium that are the
true mediators of the observed toxic effects of cobalt NPs on 3T3 fibroblasts [16].
Ion-mediated effects of NPs have been described also for zinc oxide NPs influenc-
ing the cellular processes in macrophages [17]. Other interesting examples of NP
dissolution are reported by Freitas and coworkers [18] for silver NPs of various
sizes (1040 nm) releasing ionic silver [Ag(I)] when dissolved alone in aqueous
solution or mixed with copper ions [Cu(II)] or proteins and upon reaction with the
metalloproteinase Cu(II) azurin. Particle dissolution is a similar mechanism also for
the low toxicity and biopersistence in the lung of inhaled (50 mgml1) barium
sulphate NPs [19]. The release of ions from nickel sulphate NPs (nanoballs) is
advantageously used for patch testing of nickel allergy [19]. The size [20], the
aggregation tendency [21] and the capacity to form complexes with proteins (pro-
tein corona) [19, 22] contribute to determine the biological impact of NPs. For
instance, BALB/c 3T3 fibroblasts undergo a higher degree of cell death when
exposed to cobalt NPs rather than microparticles and ions [Co(II)] [16, 23], whereas
only micro- and nanoparticles have morphological transforming potential [23].
Another study regarding the effect of cobalt NPs on gene expression indicates that
innate immunity and apoptosis are influenced, whereas microparticles and ions
affect different functional pathways [24].
Epidemiological data show that allergic reactions towards palladium, as contact
dermatitis, are increased in the last decades in people living in urban settings where
ultrafine particles (UFPs), including NPs of platinum group metals (GPE) and car-
bon, are released in the environment [25, 26].
Thus, are there scientific evidences supporting the role of nanosized material in
the development and/or exacerbation of allergic diseases? Are there contacts or
dermal sensitisers amongst nanomaterials? What are the possible mechanisms
through which a pathological outcome might derive from professional exposure?
The aim of this chapter is to describe the recent findings about these issues and
to analyse them in the perspective of occupational biosafety of nanowork.
3.2 Allergy-Related Biological Mechanisms of Nanomaterials

In Vitro
The first event for a foreign antigen to evoke an immune response consists in the
activation of the innate system cells. NPs are known to interact with these cells [27,
28], specifically devoted to getting rid of all non-self particulate matter entering the
body. These defensive players, in particular macrophages and dendritic cells, detect
and bind the NPs through molecular sensors adopted to identify bacterial and viral
pathogens called Toll-like receptors (TLRs) [29], through which they can deliver
signals in a size-dependent manner [30]. Such interaction can modify the activation
status of these cells and their fundamental activities of phagocytosis and antigen
presentation [31]. Moreover, following the interaction between NPs and the tissue-
resident cells of the innate system, including neutrophils and mast cells, an inflam-
matory process is started that leads to the production of inflammatory mediators
favouring vasodilation and chemotaxis [28]. The phagocytosis generates high levels
of reactive oxygen species (ROS) produced to eliminate xeno-(nano)particles and
linked to the pathogenetic potential of the NPs [28]. ROS induction is a critical step
in the allergic response [32]. ROS can also be produced by the metal-based NPs of
palladium and nickel for which a primary size-dependent catalytic mechanism has
been described [33].
NPs may bind to antigenic proteins and induce conformational changes exposing
cryptic epitopes (also used to obtain neo-allergens for therapeutic purposes) which,
if exposed in humans, might trigger detrimental immune responses, including
allergy [34].
A chemical substance can behave as a hapten able to form an immunogenic com-
plex with a protein carrier inducing specific antibodies; in particular, given that
crystals can only raise IgM, nanosized fullerene C60 is likely to act as such since it
evokes the production of specific IgG [35].
Preliminary data regarding the in vitro responses of human immune cells chal-
lenged with NPs are coming up: LPS-activated lymphocytes from healthy subjects
respond to the exposure to palladium NPs (510 nm) by internalising them in endo-
cytic compartments and secreting high levels of IFN- (while inhibiting the tolero-
genic cytokine IL-10) [3638] suggesting a potential role in the Th1-mediated
mechanism of delayed allergic reactions. IFN- release is also induced by
multiwalled carbon nanotubes (MWCNTs) in mitogen-stimulated T cells from
Table 3.1 List of manufactured nanomaterials and their known allergy-relevant mechanisms of
action based on in vitro studies
Manufactured
nanomaterial Cell model/species Mechanisms/effects References
Barium BaSO4 Ion release [19, 22]
sulphate Protein corona
Carbon C Human T cells (mitogen- Induction of IFN-g [29, 37]
nanotubes, stimulated)/healthy and (healthy subjects)
multiwalled allergic humans Inhibition of IL-5
(MWCNs) (allergic subjects)
Carbon C Toll-like receptor [29, 38]
nanotubes, Induction of IL-1
single-walled
(SWCNTs)
Cobalt Co 3T3 fibroblasts/ Ion release [16, 19, 22]
BALB/c mice Protein corona
Fullerene C60 C Toll-like receptors [29, 35]
Apten
Nickel oxide Ni ROS, catalytic activity [31]
Palladium Pd Peripheral blood ROS, catalytic [31, 3436]
mononuclear cells/human, activity, induction of
healthy IFN-g
Silver Ag Ion release [18]
Titanium TiO2 Peripheral blood ROS, apoptosis Personal
dioxide mononuclear cells/human data
Zinc oxide ZnO Macrophages Ion release, [17]
impairment of cell
function
healthy subjects, whereas they inhibit the allergen-induced release of IL-5 by

peripheral blood mononuclear cells (PBMCs) from mite allergic patients [39].
Titanium dioxide NPs (7 nm, anatase) induce ROS increase in human PBMCs
exposed to 25, 50 and 100 gml1 culture medium in a dose-dependent manner
after 24 and 48 h incubation; in this system, ROS induction seems to be associated
with the initiation of the programmed cell death (apoptosis) (personal unpublished
data). Cellular response to carbon nanotube exposure involves upregulation of IL-1,
participating to the intensification of the allergic inflammation [40]. Zinc oxide
NPs, the most diffused NPs in consumers goods, induce changes in the level of key
proteins controlling cell survival and cancer (p53, Ras p21 and JNKs) in ex vivo
lymphocytes isolated from lung tumours and chronic obstructive pulmonary disease
(COPD) patients, but not in those from asthmatic subjects, compared to controls [41].
Allergy-relevant mechanisms of action and effects of the manufactured NPs based
on in vitro findings are summarised in Table 3.1.
3.3 Allergy-Related Biological Mechanisms of Nanomaterials

in Animal Models
It has been shown that NPs can cause specific immune reactions, immunosuppres-
sion and autoimmunity in animal models. Mouse and rat are considered well suited
to mimic the basic immunological mechanisms of allergic sensitisation, atopic/con-
tact dermatitis and asthma development. However, the validity to predict the sensi-
tising and asthmogenic health risk for NP-exposed workers is not straightforward.
In fact, the sensitivity against NPs seems to depend on the mouse strain [42].
Furthermore, the study design is crucial to obtain relevant data from animal models.
In this regard, it seems that exposure to the test NPs should be done during the sen-
sitisation and challenge phases. Moreover, studies should focus on the use of
agglomerate/aggregate NPs. Even if not completely following such criteria and not
using standardised NPs, a consistent amount of data are emerging from animal stud-
ies regarding allergy. The exposure route seems to be relevant for the observed find-
ings: NPs cause inflammation of the lung if inhaled or instilled into the trachea or
worsen the allergic airway inflammation and act as Th2 adjuvant following pulmo-
nary exposure [43].
Some types of engineered nanomaterials induce immune responses in the lung; in
particular, they appear to alter the allergen-induced eosinophilia [44] by modulating
the balance between Th1, Th2 and Th17 cells [28].
Copper NPs are the most potent inducers of inflammation of the lung in a model
of pulmonary bacterial infection sustained by neutrophils and inflammatory cyto-
kines, an effect also caused by iron oxide NPs [45].
Adjuvant properties of NPs could have the undesirable effect of upholding aller-
gic sensitisation. For instance, 3-day long subcutaneous or inhalant administration
of carbon black NPs (22 and 39 nm) to transgenic mice (DO11.10) expressing the T
cell receptor specific for the immunodominant peptide of ovalbumin (peptide 323
339) favours the sensitisation; in fact, secondary responses, induced in peptide-
restimulated splenocytes in vitro, comprise Th2 cytokines (IL-4, IL-10 and IL-13)
and reduced expression of the transcription factor Stat4, specific for Th1 cells.
Notably, the authors find a relationship between the size of the particle and the mag-
nitude of the effect. However, in this study, equal mass of the two types of NPs were
compared implying that the total reactive surface was much higher for the smaller
ones, accounting for the higher intensity of the observed responses. Nevertheless,
carbon black NPs indeed showed a Th2 adjuvant effect in this animal model [46].
Also carbon nanofibres have IgE adjuvant capacity but are less potent than nano-
tubes in promoting allergic airway responses [47]. Engineered silica NPs act as
adjuvants enhancing allergic airway disease in mice [48].
The direct implication of NPs in the exacerbation of pre-existing type I allergy
has been shown in rats sensitised to ovalbumin. Intratracheally administered silicon
dioxide NPs (nonspherical, 1020 nm, 140180 m2g1) induced increase of IL-4 in
the lung tissue, airway remodelling and worsening of the respiratory parameters,
with more pronounced effects at the highest dose (80 gml1) [49]. Notably, such
adverse effects on lung function occur with or without ovalbumin immunisation

[49]. These findings suggest that NPs may have a direct role in the development of
bronchial asthma, not only as an exacerbating factor of a pre-existing allergic condi-
tion. Zinc oxide NPs provide an adjuvant effect to ovalbumin via a Th2 response
[50], involving TLRs and Src signalling [51], and induce eosinophilic airway
inflammation [44]. Moreover, these NPs (21 nm) do not affect oral tolerance [52].
A paradigm of the potential pro-allergic effect of nanoparticles comes from a
study on cerium dioxide NPs, used for medical applications and also present as side
products in the exhaust gas from diesel engines, able to generate multiple types of
water-soluble NPs of concern for environmental and in vivo effects. Indeed, they are
strongly suspected to promote allergic diseases since selectively taken up from alve-
olar macrophages and able to induce an inflammatory state [53].
Silver NPs have been associated with exacerbation of airway hyperactivity. Their
role in the allergic sensitisation has been studied in experimental asthma induced by
ovalbumin: the animals were exposed to 3.3 mg/ml NPs (33 nm) continuously
inhaled; in both groups of healthy and sensitised mice, these NPs caused infiltration
of neutrophils and lymphocytes in the airways (bronchocentric) and in the perito-
neum at the sites of higher accrual; moreover, it induced elevation of mediators and
effectors of the allergic response (total IgE, leukotriene E4 (LTE4), Th2 cells, IL-13
and oxidative stress) [54] and significant increase in allergen-specific IgE [55]. This
same detrimental effect on the allergic airways was associated with altered VEGF
signalling pathway and mucus hypersecretion [56]. Low-dose intratracheal instilla-
tion (0.2 mg per rat) of aggregates of nickel oxide NPs (26 nm) induces a transient
increase in chemokines associated with relentless lung inflammation and in the
expression of cytokines involved in allergy inflammation and fibrosis [57].
The case of iron oxide NPs is very interesting because the allergic response is
suppressed or enhanced depending on the particle dose: in fact, iron oxide NPs
intratracheally administered before and during ovalbumin sensitisation significantly
inhibit the allergen-specific Th2 immune response at the two highest doses (4 250,
4 500 g per mouse), whereas the lowest dose (4 100 g per mouse) promotes
the allergic response [58].
It is worth pointing out also that dermal exposure to combination of agglomer-
ates of silica NPs and mite allergens induced an IgE-biased immune response and
an increased sensitivity to anaphylaxis in not pre-sensitised mice [59].
Titanium dioxide NPs, the most produced amongst nanomaterials, are regarded
as relatively nontoxic at the concentration levels of the occupational environments
[60]. However, studies on inhalation exposure found this type of NPs to alter the
inflammatory response in asthmatic mice [61]; moreover, they aggravate atopic der-
matitis (AD)-like skin lesions in mice [62]. Lung injury-inducing high doses (32
mgkg1 twice a week for 4 weeks) of titanium dioxide NPs are not associated with
significant changes of IL-4 in the lung, ruling out the involvement of an allergic
reaction in the mechanism of damage [63]. Route-dependent immune effects are
observed following exposure to titanium dioxide-NP immune effects [64]. Also the
sensitisation status seems to determine different outcomes: ovalbumin-induced
allergic pulmonary inflammation characterised by eosinophil infiltration in the lung
and IL-4 is reduced upon inhalation of (agglomerated) titanium dioxide NPs (32
mg/ml for 6 and 42 h post-sensitisation) in asthmatic rats [65]; in non-sensitised
rats, TiO2 NPs (75 % anatase, 25 % rutile, 21 nm, aggregates of 200 nm and 2 m, 5
mgkg1) intratracheally instilled once provoke acute airway inflammation with
transitory IL-4 production, replaced by a durable Th1 response [22]. In a mouse
model, instead, the same component instilled at 550 mgkg1 (150 m aggregate
mean size) induced a Th2-mediated chronic inflammation, evidencing a species-
related bias of the immune response [66].
Polystyrene NP application to atopic dermatitis-like skin lesions (related to mite
allergen) induces local size-dependent increase of protein levels of IL-4 and various
chemokine ligands for monocytes and macrophages recruitment [67], whereas, on
normal skin, it induces sensitisation [68]. In the same AD (atopic dermatitis), an
aggravation of the pathological condition is induced size dependently by amorphous
silica NPs upon intradermal injection [69].
NPs of nickel oxide (NiO) and cobalt oxide (Co3O4) induce lung-delayed
hypersensitivity-like responses in mice [70], whereas hydroxyapatite NPs do not
produce any effect in the guinea pig model of this pathology [71]. Allergy-relevant
mechanisms of action and effects of the manufactured NPs based on in vivo findings
are summarised in Table 3.2.
Interestingly, relevant NPs in the field of advanced technologies, such as silver
NPs and fullerene C60, own intrinsic capacity useful to control the allergic reac-
tions; for instance, fullerene C60 was found to prevent the histamine release in a
mast cell-dependent anaphylaxis model [72], and silver NPs (5 nm) reduce hyper-
reactivity in a murine model of airway allergic inflammation, perhaps due to its
antioxidant and anti-inflammatory activities [73]. Topically applied zinc oxide NPs
(ZnO) suppress allergen-induced skin inflammation but induce vigorous IgE pro-
duction in the atopic dermatitis mouse model [17].
3.4 Occupational Allergic Diseases with Potential

Nanomaterial Aetiology
Taking into account the global experimental information gathered so far on the bio-
logical impact of nanomaterials, for exposed workers, the risk of developing
nanoparticle-exposure associated pathologies cannot be totally ruled out, as well as
exacerbating pre-existing pathologies. Moreover, it is conceivable that adverse
health effects may especially develop in hypersensitive populations, such as indi-
viduals with respiratory diseases (Fig. 3.1). Whatever form and chemical species
they acquire inside the body, engineered nanomaterials can induce oxidative stress
responsible for lung diseases [74] attributable to their particulate physical form.
Pleural effusion, pulmonary fibrosis and granuloma can be related to exposure to
NPs [75]. The possibility of aggravation of chemically induced occupational asthma
in the presence of NP exposure is suggested by recent findings showing that a very
Table 3.2 List of principal manufactured nanomaterials with high industrial burden and mechanisms favouring allergic diseases triggered by exposure
3
described in animal studies

Production and Biological/pathological
Nanomaterial Formula usage sectors Animal model Exposure route Mechanism outcome References
Cerium CeO2 Medical CD-1 mouse Intratracheal Release of Selective uptake by alveolar [53]
dioxide applications water-soluble macrophages
nanoparticles
Pro-inflammatory
Cobalt oxide Co3O4 Sensors, energy, Healthy rats Lung Apten Delayed-time [70]
drug delivery instillation hypersensitivity
Copper Cu Electrical, Pulmonary Inhalant Pro-inflammatory Strong lung inflammation [45]
engineering, bacterial infection sustained by neutrophils and
transport, building in mice inflammatory cytokines
and construction
Carbon black C Tyres, printer toner, Transgenic mice Intratracheal Th2 adjuvant IL-4, IL-10 and IL-13 [46]
dyes for leather or (OVA-specific T Subcutaneous Th1-specific Stat 4
textiles, cosmetics cell receptor) transcription factor
Carbon C Power, wind energy, Healthy mice Intratracheal IgE adjuvant Allergic airway disease [47]
Engineered Nanomaterials and Occupational Allergy
nanofibres aeronautical, sports,

construction,
chemical,
automotive
Iron oxide FeO Glass, paints, Healthy mice Intratracheal Neutrophil, Lung inflammation [45, 58]
coatings, inflammatory
automotive cytokines
Nickel oxide NiO Microelectronics, Healthy rats Intratracheal Pro-inflammatory Th2 cytokines [57, 70]
aerospace materials, 0.2 mg per rat chemokines
batteries, (low)
electrochromic Fibrosis
coatings and Sustained lung inflammation
materials Delayed-time hypersensitivity
35
(continued)
36

Production and Biological/pathological
Nanomaterial Formula usage sectors Animal model Exposure route Mechanism outcome References
Polystyrene (C8H8)n Atopic dermatitis Healthy mice On skin lesion Pro-inflammatory Site-dependent IL-4, [67, 68]
mouse model On normal skin chemokines
Sensitisation
Silicon SiO2 Concrete, plastics, OVA-sensitised Intratracheal Adjuvant of type Induction of IL-4 [48, 49, 59,
dioxide synthetic materials rats 140180 m2g1 I allergy 69]
Allergic airway disease (only
Healthy rats sensitised)
Adverse effect on lung
function
Silver Ag Plastics, synthetic OVA-sensitised Inhalant Th2 adjuvant Induction of IL-4, IL-13, [5456]
materials mice 7 days total IgE, allergen-specific
continuously IgE, LTE-4, Th2 cells,
neutrophils
Oxidative stress
Exacerbation of airway
hyperactivity
VEGF
Pro-inflammatory Mucus hypersecretion
Healthy mice 3.3 mgm3
Titanium TiO2 Cosmetics and paint AD mouse model Topical ROS, apoptosis Aggravation of AD [22,
dioxide Inhalant 6166]
Lung injury (IL-4 independent)
Healthy mice Intratracheal
Th1 response/Th2-mediated
Healthy rats/mice inflammation
OVA-sensitised mice
Zinc oxide ZnO Cosmetics and paint OVA-sensitised Th2 adjuvant Eosinophilic airway [44, 50,
mice inflammation 51]
AD mouse model Induction of IgE
C. Petrarca et al.
low dose (approx. 0.8 mgkg1) of intrapulmonary gold or titanium dioxide NPs
modulates a non-IgE-mediated asthma [76, 77] (Fig. 3.1). The pro-inflammatory
activity shown by most nanomaterials makes plausible the hypothesis that the occu-
pational exposure might provide stimuli able to trigger sarcoidosis-like illness
(giant cell-like nodules), typically affecting individuals exposed to foreign antigens
and to inorganic particulates and recognised and compensable as work-associated
disease [78].
Fig. 3.1 Potential influence of NPs in the development of allergic and intrinsic asthma, as sug-
gested by in vitro and in vivo findings. Pathways and molecules possibly affected by NPs activity
are coloured in red (Modified from [89])
It is well known that occupational asthma may be caused by metals (e.g. cobalt,
platinum, chromium, vanadium, nickel) and that sensitisation to the professional
agent is one of the known aetiopathogenetic mechanisms triggering this immuno-
logically mediated disease. It has been found that also nanosized metals own pro-
sensitising effects in vitro and in vivo, suggesting a potential role in the development
of this life-threatening professional disease (Fig. 3.1). The most challenging of them
regards the possibility of acting as such, as aptens or as released ions once inside the
body.
Even more concerning, a role cannot be excluded for nanoparticles in the exac-
erbation of pre-existing eosinophil-mediated allergic diseases of nonoccupational
aetiology, such as rhinitis, atopic dermatitis and food allergy, as well as other Th2-
mediated nonallergic pathologies, such as aspergillosis, eosinophilic gastroenteritis
and Churg-Strauss syndrome (Figs. 3.1 and 3.2).
Pro-sensitising effects, as well as other unpredictable immunological effects,
might be worrying for those workers of pharmaceutical industries producing nano-
Fig. 3.2 Potential role of NPs on allergen-induced airway inflammation, as sustained by in vitro
and in vivo data. Cell types, pathways and molecular mediators emerging to be influenced by NPs
are coloured in red (Modified from [89])
vaccines made of NPs loaded with immunogenic proteins to be used for immuno-
therapy or functionalised on the surface to generate immunoactivating compounds
for anticancer therapy. Several anti-allergy nanovaccines have been designed so far
with the purpose of switching off the detrimental immune response towards an aller-
gen. They consist of porous and/or polymeric substances (PLGA, dextran, silica)
with adjuvant activity (either intrinsic or conferred by LPS) that are loaded [17, 79,
80] or agglomerated [81] with the therapeutic allergen. In vitro and in vivo experi-
mental testing of these products has been carried out recently. The results suggest
beneficial effects of these nanovaccines in allergic asthma and atopic dermatitis
model that appear to rely on the potentiated adjuvancy given by the NP component.
In particular, the treatment with PLGA profilin modifies the Th2/Th1 cytokine bal-
ance and inhibits the differentiation of eosinophils [79]; dextran-NP ovalbumin acti-
vates the antigen-presenting cells and induces potent immunomodulation and
proliferative responses of allergen-specific T cells (experimentally grafted in mice)
[80]. Moreover, zinc oxide NPs, designed for local treatment of atopic dermatitis,
are able to suppress allergen-induced skin inflammation in the mouse model of this
disease but also to induce strong IgE production [17].
A very high rate of immunoactivation of bone marrow-derived dendritic cells
(MDDCs) and T cells is achieved experimentally by exposing these cells to porous
silicon nanoplatforms designed for immunotherapy, with dependency on surface
chemistry [82].
3.5 Nanoparticle Sensitising Effects in Humans
Data on the effects of occupational exposure to NPs are very limited. In fact, their
ability to induce sensitisation has been investigated for a chronic nonoccupational
exposure due to hip replacement with cobalt-chromium-coated polyethylene
NP-based prosthesis, characterised by strong metal ion release. There was a signifi-
cant increase of the two metals in the urine of the subjects who had received this
type of prosthesis, compared with those who had received the standard non-NP
type; however, no relative increase of metal allergy was observed, with a follow-up
of 5 years after surgery [83]. Even though this study does not attribute to in vivo
NPs with a higher sensitising potential compared to non-nanomaterials, this conclu-
sion is made on a limited number of subjects and a short time of observation, while
the long-term effects are not predictable [83]. Recently, a first human study of a
systemically ingested silver-NP product [84] has been carried out. Also in this case,
silver was present in serum supposed to be mostly in the ionic form, since after 14
days no evidence was found of intact silver NPs absorbed through the human diges-
tive tract or attached to blood components. Moreover, no clinically significant
changes in metabolic, haematologic, urine and physical findings, sputum morphol-
ogy and/or imaging changes were observed. Hence, even for exposed workers, ion
released from metal NPs could represent a potential allergic/health threat.
Human data are available from volunteers exposed, in research settings, to diesel
exhaust particulate (DEP) containing NPs (at >106 particlescm3, far exceeding the
worst-case exposure for workers) for whom elevated exposures have been associated
with temporary lung and systemic inflammation, thrombogenesis, vascular function
and brain activity with no evidence of a unique toxicity for NPs, as compared to
other particles, and not supporting acute toxicity of NPs (at least within DEP) by
virtue of their exclusive nanosize [85].
Although no prospective studies have still been conducted, several case reports
are already available.
In 2009, seven healthy women experienced short breath and pleural effusions
after 513 months of occupational exposure to polyacrylate NPs. These symptoms
were associated with the presence of NPs in cells and chest fluid along with lung
inflammation fibrosis and granuloma of the pleura. However, there were no findings
of allergic responses or other immunological impairments [75].
A 22-year-old student involved in work leading to synthesis of dendrimers
(nanosized particles used as drug carriers) developed contact dermatitis (erythema
multiforme-like) on his hands that progressed to involve other areas of the body.
Despite treatment with topical steroids and antihistamines, it required hospitalisa-
tion for more than 3 weeks. However, professional re-exposure caused recurrence of
the symptoms [86].
In 2014, a case report was published about a healthy 26-year-old nonsmoking
female working in an industry producing metallic inks who was assigned the task of
weighting out repeatedly 12 g of dry nickel-NP (20 nm, 4060 m2g1) powder on
the laboratory bench without wearing any personal protection. Then, she developed
throat congestion with postnasal drip and flushing of the face; concomitantly, she
manifested skin reactions to earrings and belt buckle, never observed before. The
patch test reacted positively to nickel sulphate in the standard patch testing [87].
The conclusions of a recent epidemiologic questionnaire-based study on workers
handling various types of NPs aiming at highlighting the link between the levels of
exposure and symptoms revealed sneezing as the only significant work-related
effect and allergic dermatitis as the only disease significantly worsened [88].
3.6 Discussion
It is demanding to assess whether nanomaterials can act as inducing and promoting

risk factors to safely develop nanotechnology and nanowork. This chapter repre-
sents an attempt to collect up-to-date findings and rationalise them for such purpose,
giving special focus on nanoparticle-related allergy risk. It is clear that data col-
lected so far come from heterogeneous experimental studies that are affected by a
bias due to the poor characterisation of nanomatter once it reaches the human bio-
logical matrixes. In fact, although a precise characterisation can be obtained for the
manufactured nanomaterial products, upon interaction of nanoparticles with the
organism, the newly acquired properties are virtually not predictable and rather
complex to measure (by using radioactive nanomaterials, for instance). This makes
it difficult to obtain an unequivocal assessment of a cause-effect relationship.
Moreover, in order to achieve an experimental result, high doses and short time of
exposure are applied; whereas the level of contamination is expected to be rather
low in the workplace for a chronic mode of exposure.
Lack of human data for the potential toxicity is due to two main reasons: chal-
lenge studies are not feasible in man and, in the environment, NPs are contaminated
with other numerous substances that modulate their activity. For this reason, the
workplace could represent an outstanding experimental setting because the level
and the condition of exposure are well measurable; moreover, the health surveil-
lance programme might provide the follow-up data to identify the onset and pro-
gression of any possible diseases. Researchers should take advantage of this
circumstance to carry out studies on pathogenetic mechanisms.
Being one of the epidemics of the century and one of the most prevalent diseases
in workers, allergy should be logical to carefully evaluate the risk induced by NPs
in exposed workers. Available data provide the justification for this attention. There
are studies (few) sustaining that NP exposure is associated with the appearance of
allergic pathologies, either cutaneous or IgE mediated, supported by in vitro and in
animal findings showing that various NPs are able to induce immunological modi-
fications typical of allergy. By combining health surveillance and scientific research,
it will be possible to assess nanomaterial effects on human health and prompt the
safe and sustainable production and use of nanomaterials.
Acknowledgements We thank Dr Flavia Carpiniello for the assistance in the revision of the
English language and the graphical aid.
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Chapter 4
Allergens in Occupational Allergy: Prevention
and Management Focus on Asthma
Mario Di Gioacchino, Luca Di Giampaolo, Veronica DAmbrosio,

Federica Martino, Sara Cortese, Alessia Gatta, Loredana Della Valle,
Anila Farinelli, Rocco Mangifesta, Francesco Cipollone, Qiao Niu,
and Claudia Petrarca
Abstract This article reviews the main aspects for the prevention and management
of occupational asthma due to allergic sensitization in the workplace. An accurate
allergen identification and characterization is the essential aspect of the primary
prevention. Both high and low molecular weight molecules (the first acting as
complete antigens the second as haptens) can induce asthma. Sensitization is a
dose-related phenomenon, therefore the lower the exposure the lower the risk of
sensitization. Health surveillance is the key action of the secondary prevention; it
aims at the early identification of workers with occupational exposure to asthma-
causing agents by means of respiratory questionnaires; preplacement and periodic
visits, with spirometry; immunologic tests; and further investigations to confirm
diagnosis and then remove the person from further exposure. Asthma management
M. Di Gioacchino (*)
e-mail: digioacc@unich.it
L. Di Giampaolo
Chieti, Italy
V. DAmbrosio F. Martino
Specialization School of Occupational Medicine, G. dAnnunzio University,
c/o CeSI-Met Via Colle dellAra, 66100 Chieti, Italy
S. Cortese
A. Gatta L.D. Valle A. Farinelli F. Cipollone
Specialization School of Allergy and Clinical Immunology, G. dAnnunzio University,
Chieti, Italy

DOI 10.1007/978-981-10-0351-6_4
48 M. Di Gioacchino et al.
may be identified with the tertiary prevention. In this phase other than the pharma-
cological treatment, the occupational doctor should remove the worker from the
exposure, as the reduction of allergens is a negative prognostic factor for a severe
decline of lung function. In any case, workers should be informed on the risk they
encounter at work for a better outcome of the preventive measures.
Keywords Occupational asthma Occupational allergy Work ability Removal

from the exposure Decline in pulmonary function
Occupational allergy refers to diseases or conditions caused by exposure to sub-

stances in the workplace and in whose pathogenesis allergic mechanisms (both
types I and IV) are involved [1]. Asthma, rhinitis, conjunctivitis, and dermatitis are
the most common forms. The goal of managing occupational diseases is avoiding
the onset of the diseases itself by the primary prevention, to be done after an accu-
rate allergen identification and characterization, secondary prevention whose key
action is health surveillance, and, finally, tertiary prevention with the disease man-
agement [2]. Asthma is the most severe manifestation of occupational allergy with
possible outcome in occupational disability. In this chapter we will address the vari-
ous steps for the prevention of the allergic occupational asthma.
4.1 Primary Prevention
The difficulties in preventing the onset of asthma and allergy in general in internal
medicine are all present in the occupational setting. The allergen identification is the
first issue. A careful risk assessment, through the environmental monitoring, pro-
vides fundamental information about substances encountered on the job, their con-
centration, and their sources. Such data could be used to direct the engineering
actions to be applied in order to avoid exposure to hazardous substances and to
evaluate the effectiveness of measures designed to maintain their concentration
R. Mangifesta
Immunotoxicology and Allergy Unit, CeSI, G. dAnnunzio University Foundation,
Chieti, Italy
Q. Niu
Occupational Health Department, Public Health School, Shanxi Medical University,
Shanxy, Taiyuan 030001, China
C. Petrarca
4 Allergens in Occupational Allergy 49
below the exposure limits [3]. Assessing exposure entails a scrutiny of who does
what, where, and how. However, there are frequently objective difficulties in identi-
fying the agent directly responsible for or most closely associated with the risk of
occupational allergy, as in the reported cases of occupational asthma due to allergy
to molds contaminating the cooling system of the workplace [4]. In any case, there
are few possibilities in completely preventing the onset of allergies to occupational
allergens. The main debate was to establish the workability of atopic subject who
should work in an environment rich in potential allergens. Should this subject be
employed, being atopics at risk of developing allergic diseases, including asthma?
Recent data seems to indicate that atopy should be considered insufficient to advise
against working in environment with aeroallergens, even to cross-sectional studies
have shown that the likelihood of sensitization to large molecules of biological ori-
gin [5], such as laboratory animal allergens [6], microbial enzymes, and latex glove
protein [7], is increased in atopics. On the other hand, smoking seems to be a greater
and more significant determinant risk for sensitization compared to atopy [8]. In
particular, smoking, and not atopy, appears to be a risk factor for sensitization to low
molecular weight (LMW) chemicals [9]. However, neither the atopy nor smoking is
sufficiently predictive to be used in determining the ability of a worker to participate
in a job that carries a risk of sensitization [10]. Some studies have reported that the
exclusion of atopics might still allow employment of more people who would
become sensitized than the number of those whose sensitization had been averted in
primary prevention. Therefore, evidence of atopy per se is not adequate justification
for refusing employment where there is exposure to respiratory allergens [6]. It
must be stressed that prevention of work-related diseases should rest primarily on
making the workplace safer for workers rather than by using poorly validated crite-
ria for excluding individuals from employment.
The identification of occupational allergens results mainly from reports of health
surveillance. However, there is a need for the identification of occupational aller-
gens in an earlier phase, before the onset of asthma, but no established protocols for
an efficient prospective identification of chemical respiratory sensitizers are at pres-
ent available. Recently some authors [11, 12] suggested the possibility to evaluate
the structureactivity relationship (SAR) models as potential methods to prospec-
tively conclude on the sensitization potential of LMW chemicals. However, no sin-
gle SAR model was sufficiently reliable, but combining two SARs with individual
applicability domains of the models provided reliable predictions for one-third of
the respiratory sensitizers and nonsensitizers analyzed. The authors stated that a
positive predictive value of 96 % and a negative predictive value of 89 % were
obtained. In any case for the two-thirds of the chemicals, additional information is
required (in chemico or in vitro methods) to reach a reliable conclusion.
Allergens can be complete antigens or haptens [13]. The first are high molecular
weight (HMW) organic compounds (Table 4.1) and the second LMW chemicals
that can stimulate the IgE production as haptenprotein conjugate [14]. The list of
such substances (Table 4.2) is continuously increasing.
There are many other substances, such as some metals (aluminum, vanadium),
fluxes (colophony, ethylethanolamine), insecticides (organophosphates), diisocyanates
Table 4.1 Natural allergens. Animal protein

Organic compounds of high
Fur, pelts, urine, serum
molecular weight
Insect arthropods
Mites, locust, honeybee
Plant protein
Grain, rye flour
Enzymes
Animal, plant, and microbial
Legumes
Coffee beans, soybeans
Seeds
Linseed, cottonseeds
Vegetable gums
Guar gum, acacia gum
Miscellaneous
Latex, tobacco, tea, henna
Table 4.2 Inorganic and Anhydrides

organic chemicals of low
Phthalic, trimellitic
molecular weight, known or
suspected allergens Metals
Nickel, chromium
Dyes
Paraphenylenediamine
Diisocyanates
MDI, HDI
Antibiotics
Penicillin, spiramycin
Wood dust
Red cedar, ramin
Miscellaneous
Chloramine-T, piperazine, ethylendiamine,
persulfate salts, glutaraldehyde,
a-methyldopa, triethylene, etc.)
(TDI, NDI, IDI), and others (methyl methacrylate, NO2, diesel exhaust particles, SO2,
etc.), in the work environment able to induce asthma by an IgE-independent mecha-
nism [15]. They may involve cell-mediated hypersensitivity or act through direct toxic
effect [16, 17]. These chemicals can induce the reactive airway dysfunction syndrome.
However, they may affect the immune system, and some of them seem also to favor
allergen sensitization. As example, it has been demonstrated that diesel exhaust particle
exposure results in accumulation of allergen-specific Th2/Th17 cells in the lungs,
potentiating secondary allergen recall responses and promoting the development of
allergic asthma [18].
High-level irritant exposure may cause ciliary activity decrease, massive damage
of epithelial cells, and tight junction disruption. Injured epithelial cells may activate
mucosal inflammation in particular stimulating the recruitment of inflammatory cell,

particularly eosinophils [1921]. The direct effect of pollutants on mast cells
induces inflammatory mediator and chemotactic factor release, thereby enhancing
mucosal inflammation [1922]. Some chemicals, in particular diesel exhaust parti-
cles, phthalates, and some engineered and anthropogenic nanoparticles, may also
favor allergic sensitization [23]. In particular, diesel exhaust particles enhance pro-
duction of mucosal IgE-secreting cells [20], and some toxic agents, such as lead,
chromium, platinum, and palladium (these two last produced by vehicular traffic in
the urban environment but also present in many working environments), favor the
prevalence of Th2 immune pattern of cytokines [24, 25].
Other factors besides the environmental and occupational toxic pollutants may
increase the risk of sensitization to occupational allergens. Some HLA markers were
found to be associated with sensitization to allergens as cow dander [26], isocyanates
[27], and anhydrides [28]. Genetic susceptibility to environmental exposures may
contribute to the onset of occupational diseases in the workplace [29]. For diseases
with complex and multifactorial etiology such as occupational asthma, susceptibility
studies for selected genetic polymorphisms provide additional insight into the bio-
logical mechanisms of disease. However, the value of genetic screening in occupa-
tional settings remains limited due to primarily ethical and social concerns.
The attention was also focused on the possible effects of electromagnetic fields
(ELMF) in human health [30]. The results of such studies are contrasting as some
authors reported that ELMFs favor allergic sensitization to environmental allergens
inducing a Th2 cytokine pattern [31], while others found an induction of a Th17
cytokine pattern [32].
Improving the workplace design, implementing alternative processes, changing
engineering design (extraction, containment of process, ventilation), and substitut-
ing the sensitizer with an alternative chemical, although not always feasible, are
measures useful in reducing the risk of occupational exposure. Therefore, efforts
have been made to reduce exposure to respiratory sensitizers by instituting occupa-
tional hygiene measures such as containment, improved ventilation, and (as a last
option) the use of personal protective equipment, as well as worker education to
enhance adherence to recommended measures. Examples in which one or more of
these measures have been effective include the use of latex-free materials in pre-
venting sensitization to latex [33], encapsulation of enzymes in the detergent indus-
try [34], the use of appropriate respiratory and protective devices and skin covering
to reduce the exposure to laboratory animals [35], and worker education to reduce
exposure in bakeries [36]. The most effective measures for primary prevention are a
combination of intervention in the workplace, use of protective equipment, and
worker education to the prevention itself [37]. However, the implementation of pre-
ventive measures in the workplace is suboptimal even in occupations with a well-
recognized risk of sensitization [38].
A further topic to be addressed is the value of the allergen exposure limits for the
prevention of occupational respiratory allergies. The statement of the International
Labor Organization, made in 1977, considered the exposure limits the concen-
tration in air of a harmful substance which, if the standards are respected, does not
generally have harmful effects including long-term effects on posterity on the
health of workers exposed for 810 h a day, 40 h a week; this exposure is considered
acceptable by the competent authority which determines the limits, but it is
possible that it may not completely guarantee the protection of health of all the
workers [39].
Actually, the exposure limits do not constitute an absolute dividing line between
the harmless and the harmful concentrations, but is intended solely as a guide to
prevention. In any case the exposure limits for toxic substances are not useful for
sensitizing agents. As example, sensitization to glutaraldehyde has been reported in
healthcare workers, despite its concentration in the workplace was below the expo-
sure limits [40]. However, also the development of allergic sensitization (and the
elicitation of an allergic reaction) is a threshold phenomenon. There are levels of
exposure below which sensitization will not be acquired as demonstrated by both
relevant human studies of occupational asthma and experimental models [41].
Unfortunately, although there is evidence that the acquisition of sensitization to
chemical respiratory allergens is a dose-related phenomenon and that thresholds
exist, it is frequently difficult to define accurate numerical values for threshold
exposure levels. Therefore, it is difficult to set exposure limits below exposures
regarded as safe in an absolute sense although the risk might be very low.
Moreover, it is possible that once sensitized, the airborne concentrations at which
symptoms could be provoked might be even lower than the concentrations respon-
sible for sensitization in the first instance [39].
As stated by the European Respiratory Society (ERS) task force in the guidelines
published in 2012 [42], the following measures are of high impact in controlling
work-related exposure to prevent asthma:
Exposure elimination is the strongest preventive approach to reducing the dis-
ease burden of work-related asthma and is the preferred primary prevention
approach.
If elimination is not possible, reduction is the second best option for primary
prevention of work-related asthma based on exposureresponse relationships.
There is limited evidence of the effectiveness of respirators in preventing occu-
pational asthma, and other options that are higher in the hierarchy of controls for
occupational exposures, notably eliminating or minimizing exposures at the
source or in the environment, should be used preferentially.
Do not use powdered allergen-rich natural rubber latex gloves.
Minimize skin exposure to asthma-inducing agents.
4.2 Secondary Prevention and Heath Surveillance
Health surveillance is the essence of the secondary prevention (Table 4.3). It can be
defined as the set of medical acts, aimed at protecting the health and safety of
workers, in relation to the work environment, occupational risk factors and proce-
dures for carrying out work, and at the formulation of the judgment of the specific
working task ability.
Table 4.3 Strategies for secondary prevention

Health surveillance
Replacement visit
Periodic examination
Questionnaires
Biological monitoring (Immunologic and functional tests)
Exposure assessment
Using personal protective equipment
Last line of defense in situations where control at source is clearly impracticable: man-made
exposures
Worker education
In the case of occupational asthma, secondary prevention includes early identifi-

cation of workers with occupational exposure to asthma-causing agents by means of
medical surveillance that consists of preplacement and periodic visits; respiratory
questionnaires, with spirometry; immunologic tests; and further investigations to
confirm diagnosis and then remove workers from further exposure.
Preplacement visit includes clinical, instrumental, and laboratory investigations
aimed at assessing the state of health prior to the occupational hazard exposure and
to identify any congenital or acquired abnormalities that may represent a clinical
condition of increased respiratory susceptibility toward the irritating, sensitizing,
and toxic substances, to which workers will be exposed. It has the aim to define the
ability of workers for the specific task. In occasion of the periodic medical examina-
tions, the investigations are designed to check, in the light of actual exposure condi-
tions, the onset of any early and reversible changes of the respiratory system, caused
by occupational exposures. Trends of symptom prevalence, suggesting sensitization
in relation to different categories of employees, workplaces, and tasks, should be
investigated. In this way, it is possible to control the frequency of the occupational
allergies [42], to evaluate the risk of occupational disease among the different jobs
[43], and to identify sensitizing substances [44, 45].
Health surveillance enables the early identification of adverse health effects in
individuals; it may supplement environmental monitoring in assessing control and
may contribute to the process of hazard and risk assessment; it offers information
regarding hazardous substances to which the employees are exposed, the symptoms
which may result, the potential long-term risks, and, therefore, the need to report
these symptoms to the occupational health service.
The use of questionnaires seems to be useful in detecting subjects with
occupational asthma, and such measures have been introduced in some companies
for at-risk workers employed in bakeries, working with animals, detergents,
diisocyanates, or complex platinum salts or exposed to acid anhydrides. Studies
suggest that such programs are beneficial [46, 47], although the benefit is difficult
to precisely specify. It has been suggested that the questionnaire component is likely
to be less reliable among respondents who believe that their answers might result in
job loss [48, 49].
Some occupations have a higher prevalence of asthma than other occupations. Data
of each industry should be collected with the aim of identifying worker populations
with a high burden of asthma to which disease prevention efforts should be targeted.
Anyway, striking differences are evident comparing results obtained from health sur-
veillance schemes in different countries, such as for England (SHIELD), Finland,
Chicago (SENSOR), and Quebec [50]. However, only some of those differences are
real, due to the variation on the type and size of the industries, working practices, and
environmental protection in the workplace. For example, the high prevalence of occu-
pational asthma in Finland due to animal allergens compared to the other countries
may be explained by different farming practice. On the other hand, occupational
asthma in Finland is compensated and reports are compulsory, whereas, in the other
countries, cases are based on voluntary reporting from hospital and physicians who
are unlikely to be consulted by self-employed and uninsured farmers [51].
Physicians should also consider gender differences when diagnosing and treating
asthma in working adults. In fact, among adults with work-related asthma, males
and females differ in terms of workplace exposures, occupations, and industries. In
a study on 8239 confirmed work-related asthma cases, a significant gender differ-
ence in the distribution of the disease has been shown; in fact 60 % of them were
females. Females were more likely to have work-aggravated asthma and less likely
to have new-onset asthma. Females with asthma worked in healthcare and social
assistance, educational services, retail trade industries, office and administrative
support, education training and library and as healthcare and technical practitioners.
Asthma was induced prevalently by miscellaneous chemicals, cleaning materials,
and indoor air pollutant in females and miscellaneous chemicals, mineral and inor-
ganic dusts, and pyrolysis products in males [52].
Some authors report that work-related asthma exposures are not discussed
between workers and their healthcare provider and this communication gap has
implications for asthma management [53]. In any phase the employee must be
informed on the specific risks of asthma associated with the occupation and in the
control measures to be applied.
The ERS task force guidelines (2012) for occupational asthma [46] recommend
that in performing the medical screening and surveillance, the following issues
should be performed:
Questionnaire-based identification of all workers at risk of developing work-
related asthma.
A preplacement screening in order to identify workers at higher risk of work-
related asthma.
Detection of sensitization either by specific immunoglobulin E or skin prick test.
Inform atopic subjects and subjects with preemployment sensitization about
their increased risk of work-related asthma.
In all workers with confirmed occupational rhinitis and/or nonspecific bronchial
hyperresponsiveness, periodically administer questionnaire, detect sensitization
using standardized skin prick tests or serum-specific immunoglobulin E
antibodies, and early refer symptomatic and/or sensitized subjects for special-
ized medical assessment and assessment of asthma.
Use risk stratification by diagnostic models to select exposed workers for further
medical evaluation.
Carry out exposure assessment (and all possible related interventions) simultane-
ously to the medical surveillance.
4.3 Tertiary Prevention and Disease Management
Despite applying primary prevention measures and a proper health surveillance,

asthma will affect some workers. Literature reports that the population attributable
risk of occupational asthma is between 10 and 25 %, equivalent to an incidence of
250300 cases per one million people per year [54]. Individual cases need to be
carefully investigated and managed. The case management includes the disease
monitoring with symptom score, inflammatory and immune biomarkers, and func-
tional parameters (Table 4.4). Among immune biomarkers the usefulness of specific
IgE in detecting allergies to organic compounds of HMW is well known. Their use-
ful role has also been reported in allergy to LMW [55]. It has been shown that spe-
cific IgE to isocyanates and several other LMW chemicals is more specific than
sensitive index of occupational sensitization, and their sensitivity is lower when
assayed after cessation of the exposure, with a half-life of 12 years [56]. Therefore,
specific IgE have a valuable role to play not only in the diagnosis but also in the
surveillance of exposed workers, since the persistence of high specific IgE levels
may indicate the persistence of exposure [56].
Eosinophil cationic protein (ECP) is a relevant biomarker of eosinophilic inflam-
mation in asthmatic patients [57, 58], and the eosinophilic inflammation is associ-
ated with a great decline of FEV1 [59]. However, rather than single determinations,
the increase in serum ECP between pre- and post- allergen exposures has a strong
correlation with clinical parameters and nonspecific bronchial hyperreactivity [60].
Nonspecific bronchial hyperactivity is indeed an early and sensitive marker of
bronchial response to occupational allergens. An increase in nonspecific bronchial
hyperreactivity has been found in patients with possible occupational asthma in
which the specific bronchial challenge was negative [61].
Table 4.4 Strategies for Disease monitoring

tertiary prevention
Biomarkers, PEF, etc.
Treatment of the disease
Guidelines for asthma treatment
Removal of the exposure
As early as possible
Reduction of the exposure
Less effective then removal
Reporting and the consideration of
workers compensation while off work
The optimal management of the occupational asthma and allergies in general is

the removal of affected workers from the exposure, which in practice may be diffi-
cult to achieve. It includes avoidance of certain tasks and exposures and relocation
to other areas or processes within the workplace. Recent papers in literature com-
pared the effectiveness of complete removal from exposure to the causative agent
with reduction of exposure and continued exposure in the management of occupa-
tional asthma. Results suggested that complete removal from exposure resulted in
the best outcome in terms of symptoms, lung function, and airway hyperresponsive-
ness. Reduction of exposure appeared to be less effective in terms of improving
asthma but was also less likely to result in loss of income or unemployment [62].
Some authors reported that a reduction of exposure was associated with a lower
likelihood of improvement and recovery of asthma symptoms and a higher risk of
worsening of the symptoms and nonspecific bronchial hyperresponsiveness, com-
pared with complete avoidance of exposure [63]. The decline in pulmonary function
in asthmatic workers is particularly severe in those unable to avoid allergen expo-
sure. In fact, it has been reported that, despite an optimal pharmacological therapy,
the pulmonary function decay slope was steeper in workers continuously exposed to
the sensitizing agent (even at reduced level) than in those with a complete cessation
of exposure: final FEV1 loss was 512.5 180 ml versus 332.5 108 ml, respec-
tively. The difference became significant after 4 years from the cessation of the
exposure. The study shows that the cessation of the exposure to allergen in the
workplace appears the most effective measure in limiting pulmonary function
decline in asthmatic workers and underlines the importance of allergic risk assess-
ment and control in the management of occupational asthma [64].
Critical analysis of available evidence indicates the following: 1) persistent
exposure to the causal agent is more likely to result in asthma worsening than com-
plete avoidance; (2) there is insufficient evidence to determine whether pharmaco-
logical treatment can alter the course of asthma in subjects who remain exposed; (3)
avoidance of exposure leads to recovery of asthma in less than one-third of affected
workers; (4) reduction of exposure seems to be less beneficial than complete avoid-
ance of exposure; and (5) personal respiratory equipment does not provide complete
protection [65].
In some cases the causative allergen-inducing asthma is present also outside the
workplace and this situation represents a negative prognostic factor. This is the case
of latex that affects a large population of healthcare workers [66, 67]. In the occupa-
tional setting, latex gloves and other latex-made equipment have been replaced with
powder-free latex or nitrile gloves of latex-free compounds with a reduction of
latex-induced diseases including asthma [33]. However, although there was
improvement after implementation of powder-free latex gloves, there are still a con-
siderable number of healthcare workers with latex-related asthma symptoms, likely
due to nonoccupational latex exposure [68].
Recommendations made by the ERS task force on the management of work-
related asthma [42] suggest that:
Patients, physicians, and employers should be informed that persistence of expo-

sure to the causal agent is likely to result in a deterioration of asthma symptoms
and airway obstruction.
Patients and their attending physicians should be aware that complete avoidance
of exposure is associated with the highest probability of improvement, but may
not lead to a complete recovery from asthma.
Reduction of exposure to the causal agent can be considered an alternative to
complete avoidance, but this approach requires careful medical monitoring in
order to ensure an early identification of asthma worsening.
The use of respiratory protective equipment should not be regarded as a safe
approach, especially in the long term and in patients with severe asthma.
Anti-asthma medications should not be regarded as a reasonable alternative to
environmental interventions.
A further possibility to treat occupational asthma has recently been introduced
with the specific immunotherapy that has been tested for a few occupational sensi-
tizing agents with IgE-dependent reactions [69], in particular healthcare workers
who were allergic to latex [70] but also in small number of workers who were aller-
gic to cereal [71], sea squirt (Hoya asthma) [72], and laboratory animals [73]. The
authors reported encouraging results, but whether asthma outcomes are altered in
the long term remains to be determined, and further studies are needed before
immunotherapy can be recommended.
Also the use of anti-IgE has shown promising results particularly in severe asth-
matic workers. During treatment, nine of the ten treated patients exhibited a lower
rate of asthma exacerbations and used less oral or inhaled corticosteroids. Seven
patients were able to continue working at the same workplace as before treatment
[74]. However, as for the immunotherapy, also anti-IgE requires further prospective
studies to be introduced as routine treatment in occupational asthma.
The last resort of the tertiary prevention is the removal of the patient entirely
from the workplace. Unfortunately, removal from the exposure is not always associ-
ated with a complete recovery from the disease [75, 76]. Table 4.5 reports the
negative prognostic factor for the persistence of bronchial obstruction after the
removal from the exposure [7779].
Table 4.5 Negative prognostic factor for the persistence of bronchial obstruction after the removal
from the exposure in workers affected by occupational asthma
Type of causal allergen, in particular LMW substances (such as diisocyanates, colophony
fumes, and platinum salts)
Long duration of symptoms before diagnosis
Severity of symptoms and airway obstruction at the time of cessation of exposure
Dual response after specific challenge test
Persistence of markers of inflammation in BAL and in induced sputum
Prior history of wheezing or of smoking
Employees suffering from an occupational asthma are often retired or dismissed

on medical grounds. The society should assist the patient with a workers com-
pensation claim when applicable, to limit the socioeconomic effects of the
diagnosis.
In this case, two specific questions need to be answered with regard to such a
decision on grounds of occupational asthma:
Is the employee not well enough in terms of physical capacity to perform work,
and so likely to remain unwell that no other job is possible for that particular
employee?
Second, are all the possible jobs so unsafe, because of exposure to the sensitizer
or to nonspecific irritants, that this employee could not undertake any such jobs
without significant risks to his health and safety?
Of course all efforts for therapeutic management of the suffering worker are to
be pursued in any case.
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Chapter 5
Particulate-Driven Type-2 Immunity
and Allergic Responses
Etsushi Kuroda, Burcu Temizoz, Cevayir Coban, Koji Ozasa, and Ken J. Ishii
Abstract It is thought that particle pollutants exacerbate allergic responses that are
defined as highly increased airway cellular responses and IgE production. These
particulates are nanometer- to micrometer-sized particles such as particulate matter
2.5 (PM2.5), diesel exhaust particles, and Asian sand dust (ASD). Other than par-
ticle pollutants, particulates with similar sizes such as aluminum salts (alum) and
silica are also known to induce type-2 immune responses that are characterized by
the activation of eosinophils and the induction of antigen-specific serum IgE in vivo.
All of these particulates have a common feature in that they function as adjuvants
and promote antigen-specific immune responses. An adjuvant is an activator of
innate immunity, and innate immunity is thought to be required for the adequate
induction of adaptive immunity. In general, innate immune cells are activated
through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and
E. Kuroda (*) K.J. Ishii (*)

Laboratory of Vaccine Science, WPI Immunology Frontier Research Center (IFReC),
Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Laboratory of Vaccine Design, Center for Drug Design Research, National Institute
of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan
Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation,
Health and Nutrition (NIBIOHN), Osaka, Japan
e-mail: kuroetu@ifrec.osaka-u.ac.jp; kenishii@biken.osaka-u.ac.jp
B. Temizoz
Laboratory of Vaccine Science, WPI Immunology Frontier Research Center (IFReC),
C. Coban
Laboratory of Malaria Immunology, WPI Immunology Frontier Research Center (IFReC),
K. Ozasa
Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation,
Health and Nutrition (NIBIOHN), Osaka 567-0085, Japan
Department of Pediatrics, Yokohama City University Graduate School of Medicine,
Yokohama, Kanagawa 236-0004, Japan

DOI 10.1007/978-981-10-0351-6_5
64 E. Kuroda et al.
induce inflammatory responses. However, the basis for the adjuvanticity of these
particulates including particle pollutants and the mechanisms by which they elicit
allergic responses are still unclear. In addition, not all particulate adjuvants induce
allergic responses. In this review, we will discuss the proposed mechanisms behind
the activity of particulate adjuvants and their role in exacerbation of allergic
inflammation.
Keywords Particulates Adjuvants Type-2 immunity IgE Damage-associated

molecular patterns Innate immunity
Abbreviations
ASC Apoptosis-associated speck-like protein containing a caspase recruit-

ment domain
ASD Asian sand dust
CGAMP Cyclic GMP-AMP
CGAS Cyclic GMP-AMP synthase
CLR C-type lectin receptor
CNT Carbon nanotube
DAMP Damage-associated molecular pattern
DC Dendritic cell
DEP Diesel exhaust particle
GC Germinal center
ILC2 Group 2 innate lymphoid cell
IRF Interferon regulatory factor
KO Knockout
MSU Monosodium urate
NKT cell Natural killer T cell
OVA Ovalbumin
PAMP Pathogen-associated molecular pattern
PG Prostaglandin
PGLA Poly(lactide-co-glycolide)
PM2.5 Particulate matter 2.5
PRR Pattern recognition receptor
PTGS PGE synthase
STAT Signal transducer and activator of transcription
STING Stimulator of interferon genes
Syk Spleen tyrosine kinase
TBK TANK-binding kinase
Tfh cell Follicular helper T cell
TLR Toll-like receptor
WT Wild type
5 Particulate-Driven Type-2 Immunity and Allergic Responses 65
5.1 Introduction
Recently, the number of patients suffering from allergic disease such as asthma or
rhinitis has increased especially in developed countries. The reason for this increase
remains unclear, but many studies have demonstrated that particle pollutants such as
diesel exhaust particles (DEPs) and ASD may be involved in the exacerbation of
allergic responses [18]. In fact, epidemiological studies have reported that increased
particulate matter in air was significantly associated with increased asthma hospital-
ization [9, 10]. Allergic responses are mediated by type-2 immune responses, char-
acterized by activation of eosinophils and induction of antigen-specific serum IgE
in vivo [11]. Some particulates and crystals, including particle pollutants, are known
to stimulate immune responses to induce and enhance type-2 immune responses
[1217]. These particulates are thought to function as adjuvants; for example, alum
is a recognized and widely used particulate adjuvant [1820]. Alum has been used
as a human vaccine adjuvant for more than 80 years; however, the detailed mecha-
nism of action of alum is yet to be determined. Thus, the basis for the adjuvanticity
of these particulates including particle pollutants and the mechanisms by which
they elicit allergic responses remain poorly understood.
5.2 Innate Immunity and Adaptive Immunity
Immune responses are categorized into two types: innate and adaptive [21, 22].
Macrophage and dendritic cells (DCs) are major players in innate immunity. They
engulf pathogens or antigens as the first line of defense and then transmit informa-
tion to the adaptive immune systems via antigen presentation. Adaptive immunity is
mediated by T cells, B cells, and memory cells, which all contribute to antigen-
specific immune responses. Interestingly, recent studies have demonstrated that
innate immunity is required for the adequate induction of adaptive immunity [21
23]. In general, innate immune cells are activated by pathogens or pathogen-derived
factors (e.g., pathogen-associated molecular patterns or PAMPs) through PRRs,
such as TLRs. Then PRRs transduce activating signals into cells and induce adap-
tive immunity (Fig. 5.1) [2427]. In addition to PAMPs, factors released by cell
death (damage-associated molecular patterns or DAMPs) also activate PRRs on
innate cells to induce inflammatory responses. Lipids, sugars, metabolites, nucleic
acids, and cytokines are all categorized as DAMPs, and activation of innate immu-
nity by DAMPs is considered to be critical for adjuvant activity [2836]. Thus, both
exogenous and endogenous factors act as adjuvants activating innate immune cells
and inducing adaptive responses.
66 E. Kuroda et al.
Innate Immunity Adaptive Immunity

Pathogens Macrophages
(virus/bacteria) Dendritic cells Th1/Th2 cells
. c ytokine
. an1gen
PRRs presenta1on B cells IgG
(TLR etc)
Macrophages
Phagocytosis? Dendritic cells cytokine
Th2 cells
PRRs? an1gen
presenta1on
other unique B cells IgE
Particulates causing type-2 responses mechanisms?
(alum, PM2.5, sand dust)
allergic responses
Fig. 5.1 Innate immunity is required for the induction of adaptive immunity. Innate cells sense
pathogen via PRRs such as TLR, and then PRRs transduce activating signals into cells. Activated
innate cells induce inflammatory responses and promote the activation of adaptive immunity.
However, mechanism of action of particulate adjuvant remains poorly understood
5.3 Particulate-Induced Immune Responses
Many studies have reported the adjuvant activity of particulate matter and particulate-
induced allergic inflammation [18, 1217]. Representative examples of immune
responses induced by particulate matter are presented below.
5.3.1 Particle Pollutants
The most well-known particle pollutant is DEPs. In a mouse model, DEPs func-
tioned as adjuvant and induced ovalbumin (OVA)-specific IgE responses, infiltra-
tion of eosinophils, and goblet cell hyperplasia [5, 7]. Similar to DEP, ASD, an
aerosol from central and northwestern China that is considered to affect respiratory
health, has also been reported to induce allergic inflammation in the lung. Like
DEPs, ASD is thought to function as an adjuvant inducing allergen (OVA)-specific
responses [3, 4]. A recent study demonstrated that particulate matter 2.5 (PM2.5 is
defined as having a particulate size of around or less than 2.5 m in diameter), pres-
ent in air, exacerbated allergic lung inflammation in OVA-sensitized and challenged
asthmatic mice [37]. In addition, another study showed that intranasal administra-
tion of PM2.5 exacerbated allergic lung inflammation in allergy-prone NC/Nga
mice [38]. These results suggested that PM2.5 may increase the risk of allergic
asthma in allergy-prone children.
5.3.2 Inorganic Particles
Many studies have shown that metal-, chemical-, and mineral-derived particulates
have strong adjuvant activity. The most well-known inorganic particulate for immu-
nologists is alum. In general, alum adjuvant preferentially activates humoral immu-
nity to induce antigen-specific antibody responses [1820]. However, alum is
frequently used to generate mouse models of allergic diseases because it strongly
induces type-2 immune responses. Similar to alum, crystalline silica is also reported
to induce antigen-specific IgE and IgG1 in a mouse model [15]. A recent study
demonstrated that inorganic crystalline materials consisting of heterogeneous layers
of double hydroxides, such as lithium aluminum, calcium aluminum, and magne-
sium aluminum, have strong adjuvant activities and induce high levels of antigen-
specific IgE [39]. Nickel is a representative allergen that induces contact dermatitis,
and nickel oxide nanoparticles also function as type-2 immune adjuvants inducing
IgE [14].
5.3.3 Biological Particulates and Metabolites
Several particulates and crystals are generated in the body and induce immune
responses through their adjuvanticity. Monosodium urate (MSU) crystals are gener-
ated from saturating concentration of uric acid and are the causative agent of gout.
Since uric acid is released from damaged cells, MSU crystals are thought to be
DAMPs and exhibit strong type-2 adjuvant activity [12, 34, 4042]. Recent studies
demonstrated that MSU crystals play a pivotal role in house dust mite antigen-
induced allergic lung inflammation [43]. Chitin is a component of the cell wall of
fungi, helminth, and insects. Chitin particles, which are biopolymers of N-acetyl-D-
glucosamine, can elicit type-2 immune responses through the induction of interleu-
kin (IL)-4 from eosinophils and basophils [17].
5.3.4 Artificial Particulates
Carbon nanotubes (CNTs) are a well-known carbon nanomaterial with a cylindrical

structure that have been applied for use in drug delivery and as semiconductors.
Several studies have indicated that CNTs induce lung inflammation [44, 45]. In
addition, recent studies have demonstrated that subcutaneous, intranasal, or intratra-
cheal instillation of CNTs plus OVA significantly induced OVA-specific IgE in
serum and infiltration of eosinophils in the lung. As with other particulates, CNTs
function as type-2 adjuvants to induce antigen-specific Th2 responses [6, 8]. It is not
only cylindrical carbon structures that display adjuvant activity; carbon black,
68 E. Kuroda et al.
a colloidal carbon particle, also possesses adjuvant activity and particularly in ultra-
fine form enhanced antigen-specific IgE and induced allergic airway inflammation
[4648].
5.3.5 Particulate Adjuvant Not Causing Allergic Inflammation
Induction of type-2 responses is one of the features of particulate adjuvant; however

not all particulates induce allergic inflammation. The biocrystalline substance
hemozoin is a hemin detoxification by-product of malaria parasites. Hemozoin and
synthetic hemozoin (also known as -hematin) that do not induce allergic inflamma-
tion display strong adjuvant activity [49, 50]. Recently, synthetic hemozoin was
tested as a potential influenza vaccine adjuvant [51, 52]. Poly(lactide-co-glycolide)
(PGLA) is a biodegradable polymeric nanoparticle that initially attracted attention
as a potent delivery system [53]. However, this particle also displays adjuvant activ-
ity inducing antigen-specific IgG1 and IgG2c [53, 54]. Interestingly, PGLA stimu-
lates innate cells such as DCs to produce IL-1 and IL-1 through PRR activation
[54]. Recently, we reported that hydroxyapatite nanoparticles function as a vaccine
adjuvant [55]. The hydroxyapatite particle is a less-toxic particulate adjuvant due to
its characteristic of biodegradation and is a promising influenza vaccine adjuvant,
similar to PGLA.
Thus, not all but most of the particulates described above function as adjuvants
to enhance antigen (allergen)-specific type-2 immune responses. Therefore, it has
been hypothesized that particulate adjuvants causing allergy evoke similar and
unique signals in innate cells to preferentially activate type-2 immune responses.
5.4 Molecular Mechanisms of Action of Particulate

Adjuvants Causing Type-2 Immunity
Many scientists have investigated the mechanisms of action of particulate adju-

vants; however, detailed elucidation is yet to be reported. Many potential mecha-
nisms have been proposed, particularly focusing on the activation of innate immune
responses by particulate adjuvants. The findings of these studies are detailed below.
5.4.1 TLR and MyD88 Signaling
TLR and MyD88 signaling is one of the major signaling pathways for the activation
of innate cells such as macrophages and DCs. Schnare et al. investigated the effect
of the particulate adjuvant alum on IgG and IgE responses in MyD88-deficient
mice [56]. MyD88-deficient mice naturally display excess quantities of total IgE,
because T cells from knockout (KO) mice release increased amounts of IL-13.
However, antigen-specific IgE responses were comparable between wild-type (WT)

and KO mice that were immunized with alum plus OVA by footpad injection [56].
Furthermore, Gavin et al. also investigated alum adjuvanticity using MyD88 and
Toll/IL-1 receptor domain-coding adaptor inducing IFN- (TRIF) double-KO mice,
which completely lack TLR signaling. Similarly to MyD88 deficiency, the antibody
responses were comparable between WT and double-KO mice immunized with
trinitrophenol-hemocyanin plus alum by intraperitoneal injection [57]. However,
Matsushita et al. demonstrated that B-cell-specific MyD88-deficient mice displayed
reduced levels of serum antigen-specific IgE and IgG1 after the immunization with
alum plus OVA through direct administration to the airway [58]. Thus, the require-
ment for MyD88 signaling might be dependent on the administration route and the
specific cell type.
Several studies also demonstrated the requirement of MyD88 signaling in ambi-
ent air pollution particle-induced allergic responses. Becker et al. showed that
microbial components on ambient particulate matter stimulate alveolar macrophage
to produce inflammatory cytokines in a TLR4-dependent manner [59]. In addition,
Ichinose et al. reported that microbial materials, in particular TLR2 ligands such as
-glucan and peptidoglycan, are involved in ASD-induced lung eosinophilia. ASD,
to which LPS and -glucan adhere, induced eosinophil infiltration in the lung; how-
ever this was reduced after heating of the ASD to exclude microbial components
[60, 61]. In fact, TLR ligands are known to induce allergic lung inflammation [62],
suggesting that ambient particulate adjuvant activates type-2 immune responses, in
part, through TLR and MyD88 signaling promoted by microbial components
adhered to the particulate.
Natural and synthetic hemozoin crystals that are not particulate adjuvants caus-
ing allergy also seem to activate MyD88 signaling which is required for adjuvant
activity. The underlying mechanisms of the adjuvant activity of hemozoin remain to
be investigated [49].
5.4.2 Inflammasome
The inflammasome is a type of intracellular PRR that is categorized into the NOD-
like receptors. There are four classes of inflammasome, NLRP1, NLRP3, NLRC4
(IPAF), and AIM2, of which NLRP3 is the best characterized [24]. Upon activation,
NLRP3 forms a multiprotein complex with apoptosis-associated speck-like protein
containing a caspase recruitment domain (ASC) and caspase-1. This complex pro-
motes the secretion of the pro-inflammatory cytokines IL-1 and IL-18 through the
action of caspase-1 [24]. Initially, it was demonstrated that the NLRP3 inflamma-
some is activated by invasive infection and induces inflammatory responses [24].
Later in 2008, several reports demonstrated that particulates or crystals, such as
silica, alum, and asbestos, stimulate macrophages and DCs to produce IL-1 and
IL-18 through the activation of the NLRP3 inflammasome [6365]. The NLRP3
inflammasome has been reported to be involved in type-2 immune responses, and in
addition, alum-induced type-2 responses, especially IgG1 and IgE responses, are
70 E. Kuroda et al.
significantly reduced in NLRP3-, ASC-, and caspase-1-deficient mice. Interestingly,

most particulate adjuvants activate the NLRP3 inflammasome in macrophages and
DCs to produce mature IL-1. For example, in addition to alum and silica, MSU
acts as activator of the NLRP3 inflammasome [66]. Ambient particle pollutants,
such as DEP, ASD, and PM2.5, are reported to stimulate innate cells to produce
IL-1 and IL-18, implicating the importance of inflammasome activation [38, 67,
68]. In addition, PGLA and hydroxyapatite nanoparticles that do not induce allergic
responses function as a vaccine adjuvant and induce IL-1 through the NLRP3
inflammasome [54, 55]. However, other reports have shown that the NLRP3 inflam-
masome is dispensable for adjuvant activity of particulates. In addition, it has been
reported that the NLRP3 inflammasome is not required for antigen-specific anti-
body production induced by sensitization with alum plus antigens [6971]. These
contradictory reports on the role of the NLRP3 inflammasome indicate that further
investigation is required to determine the importance of the inflammasome in par-
ticulate adjuvanticity. Inflammasome activation induced by particulates seems to be
important for the induction of acute inflammation in the lung. Furthermore, several
studies have shown that most inflammasome activators also induce IL-1 release
from DCs, and particulate adjuvants such as alum, silica, MSU, and TiO2 stimulate
DCs to release IL-1, a process that is partially dependent on the NLRP3 inflamma-
some [72, 73]. Another study also reported that IL-1 release was partially reduced
in response to PGLA and polystyrene microparticles in DCs from NLRP3-deficient
mice [54]. The detailed mechanism of inflammasome-dependent IL-1 release by
particulate adjuvant is still unclear. Interestingly, a recent study demonstrated that
IL-1 and IL-1 control inflammation at different stages, i.e., initiation and mainte-
nance [74]. Thus, particulate adjuvants may regulate a many-sided immune response
through NLRP3 inflammasome activation during inflammation. Recent reports have
demonstrated a novel mechanism of inflammasome to perpetuate inflammatory
responses [75, 76]. After the activation of inflammasome, ASC assembles into a
large protein aggregate with NLRP3 and caspase-1. The inflammasome particles, or
specks, are released during pyroptotic cell death and taken up by surrounding
phagocyte to lead to inflammation through the release of IL-1. Thus, ASC speck
acts as danger signals to spread inflammatory responses.
5.4.3 MSU Crystals
Uric acid is an intracellular purine catabolite that is released from dying or stressed
cells. At saturated concentrations of uric acid, MSU crystals are formed. MSU crys-
tals are considered as DAMPs that regulate immune responses at the site of inflam-
mation, such as those that occur during gout. In 2003, Shi et al. demonstrated that
uric acid and its crystals induce DC maturation and activation [77]. Interestingly,
MSU crystals are reported to preferentially induce type-2 immune responses
through their adjuvant activity. Kool et al. reported that levels of uric acid are
increased in the peritoneal cavity after i.p. injection of alum. In addition, released
uric acid seems to induce antigen-specific T-cell responses because administration
of uricase significantly reduced the activation of T cells [12, 40]. Some particulates,
such as alum and silica, are known to induce cell death. Furthermore, it is reported
that ambient particle pollutants, PM2.5 and DEP, and metal-based nanoparticles
also showed cytotoxic activity on macrophages and monocytes through the produc-
tion of reactive oxygen species [7880]. Given that particulates induce cell death,
uric acid and MSU crystals released from dying cells by particulates might contrib-
ute to allergic inflammation. In fact, a recent study showed that allergic lung inflam-
mation induced by the administration of airborne allergen to the airway is a uric
acid- and IL-33-dependent mechanism and treatment with an inhibitor of uric acid
synthesis attenuated the onset of asthma [43]. Kool et al. demonstrated that uric
acid-primed inflammatory monocytes and DCs participate in T-cell activation
through IL-1 and MyD88 signaling [40]. In addition, since uric acid (and its crystal
form) is known to induce IL-1 through the activation of the NLRP3 inflammasome
as described above, this adjuvant effect was considered to be dependent on IL-1
released by the activated inflammasome. However, it was reported that the NLRP3
inflammasome, IL-1, and MyD88 signaling are dispensable for the adjuvanticity of
uric acid induced by alum [12, 14]. In addition, it was shown that spleen tyrosine
kinase (Syk) and PI3-kinase in inflammatory monocytes and DCs participate in
type-2 immune responses induced by uric acid [12]. So far, several papers have
shown the importance of Syk in activation of macrophages and DCs. The underly-
ing mechanisms of Syk activation by particulates and the role of Syk in type-2
immune responses are interesting issues that require further investigation [12, 14,
81]. Recent studies have reported the importance of Syk in immune responses
induced by dead cells, demonstrating that Clec9a, one of the C-type lectin receptors
(CLRs) in PRRs, senses necrotic cells and induces DC activation through the Syk-
dependent signaling pathway [82, 83]. Several CLRs are coupled with Syk and
transduce activating signals into cells [27]. Clec9a recognizes F-actin expressed on
necrotic cells [82, 83]. Thus, Syk may play an important role in signal transduction
for factors from dying cells.
The recognition mechanisms of MSU crystals have also been investigated. Ng
et al. demonstrated that MSU crystals interact with the lipid raft on DCs in a
receptor-independent manner. This interaction causes lipid sorting and transduces a
signal into cells that then leads to recruitment and activation of Syk [81]. Flach et al.
reported that alum also binds to the lipid raft on DCs and then activates Syk and
PI3-kinase, similarly to MSU crystals. Then DCs activate T cells through the inter-
action with intracellular adhesion molecule (ICAM)-1 and leukocyte function-
associated antigen (LFA)-1 [84].
5.4.4 Prostaglandin Production
Previously, we reported the unique function of particulates in eliciting type-2

immune responses. We focused on the particulates alum and silica and found that
these particulates stimulated macrophages and DCs to produce the lipid mediator
prostaglandin (PG), similar to the activation of the NLRP3 inflammasome [14].
72 E. Kuroda et al.
PGE2, a well-characterized pro-inflammatory lipid mediator, is an arachidonic acid

metabolite that is mainly produced by myeloid-lineage cells such as DCs and mac-
rophages [85]. PGE2 performs various functions in the regulation of immune
responses, one of which is the suppression of type-1 immune responses by inhibit-
ing the production of type-1 cytokines such as IFN- and IL-12 [8688].
Alum and silica stimulate LPS-primed macrophages and DCs to activate the
NLRP3 inflammasome and release IL-1 and IL-18; in addition, we also observed
PGE2 release from particulate-activated macrophages and DCs. PGE2 release was
shown to be independent of the NLRP3 inflammasome and IL-1 signaling because
inflammasome-deficient macrophages, such as NLRP3 KO, ASC KO, or caspase-1
KO macrophages, still produced normal levels of PGE2 in response to alum and
silica. Interestingly, PGE2 production in response to alum and silica was mediated
by the activation of Syk, which is an important molecule for the activation of DCs
by MSU crystals. To clarify the role of PGE2 in immune responses in vivo, we used
PGE synthase (PTGES)-deficient mice [89]. Macrophages from PTGES-deficient
mice did not produce PGE2 in response to alum or silica. In addition, PTGES-
deficient mice displayed reduced levels of antigen-specific IgE after immunization
with alum + OVA or silica + OVA, indicating that particulate-induced PGE2 regu-
lates IgE production in vivo. Previous studies have shown that PGE2 facilitates IgE
production from B cells stimulated with IL-4, through the intracellular accumula-
tion of cAMP [90, 91]. Furthermore, a recent study demonstrated that the elevated
levels of PGE2 in the gut are involved in allergic airway inflammation by the altera-
tion of macrophage phenotypes toward type-2 macrophage (M2) [92].
We also found that many particulates such as MSU crystals, PGLA, nickel oxide,
amorphous silica, and CNTs also stimulate macrophages to produce inflammasome-
dependent IL-1 and inflammasome-independent PGE2. In addition, increased
amounts of PGE2 release were observed in dying cells, indicating that, as with uric
acid release, PGE2 release is an indicator of cell death and might function as a
DAMP (Kuroda et al. unpublished data). These results suggest that PGE2 release in
response to particulates is one of the markers of the adjuvanticity of particulates and
may be a potent inducer of allergic inflammation.
5.4.5 Nucleic Acid
As described above, DAMP release in response to a particulate seems to be required

for the induction of immune responses [28]. Recent studies have shown that a chem-
ical agent that has cytotoxic activity acts as an adjuvant to induce IgE production,
suggesting that particulate-induced DAMPs may participate in allergic inflamma-
tion [93, 94]. DNA released from host cells appears to be a DAMP. Marichal et al.
demonstrated that DNA from damaged host cells is involved in the adjuvanticity of
alum [16]. This study showed that host DNA is released from damaged phagocytes
such as macrophage and neutrophils at the site of alum injection. The released DNA
functions as an adjuvant and induces antigen-specific IgG and IgE responses.

Interestingly, treatment with DNase I at the injection site of alum + OVA signifi-
cantly reduced OVA-specific antibody responses. In addition, purified genomic
DNA mixed with antigen mimicked the adjuvant activity of alum and induced
antigen-specific antibody responses. This finding indicates that DNA released from
host cells is a critical factor for the activation of innate and adaptive immune
responses. TANK-binding kinase 1(TBK-1) and interferon regulatory factor 3 (IRF-
3) are reported to be important signaling molecules downstream of specific recep-
tors for DNA recognition in innate cells [95]. This study demonstrated the attenuated
adjuvant activities of alum or host DNA in TBK1- or IRF3-deficient mice, with IgE
responses being markedly reduced. In addition, IRF3 participates in the recruitment
of inflammatory monocytes and DCs, cells responsible for the induction of type-2
immune responses, suggesting that IRF3 regulates the type-2 adjuvant activity of
alum through the signaling pathway for host DNA recognition during an innate
immune response. The IRF3-mediated activation of inflammatory DCs was con-
trolled by IL-12p80 release, a p40 homodimer. Furthermore, treatment of IL-12p80
neutralizing antibody partially reduced IgE responses in mice immunized with alum
+ OVA. Given that many particulates with adjuvant activity are known to induce cell
death, the adjuvant activity of particulates is considered to be closely linked to cell
death and consequently host DNA release. CpG oligodeoxynucleotide is a well-
known DNA-based adjuvant that is recognized by TLR9, but it is reported to be a
strong inducer of type-1 immunity unlike the DNA released from dying cells [96,
97], suggesting that different types of DNA induce type-2 immune responses
through signal transduction pathways other than TLR9 signaling.
A recent study demonstrated that DNA released from damaged cells directly
stimulates naive CD4+ T cells to induce Th2 differentiation through an unknown
DNA sensor and might be involved in allergic inflammation [98]. The underlying
mechanisms for Th2 differentiation were mediated by the downregulation of T-bet
and the upregulation of GATA-3 expression. Furthermore, a recently identified
DNA sensor, cyclic GMP-AMP synthase (cGAS), might be important for type-2
immunity [99]. Activated cGAS induces generation of cyclic AMP-GMP (cGAMP),
and this cyclic nucleotide functions as the ligand for the ER-resident adaptor mol-
ecules, stimulator of interferon genes (STING). Activated STING is known to regu-
late the TBK1-IRF3 signaling pathway to induce type-1 IFN production [100, 101].
Our previous studies demonstrated that cGAMP and synthetic STING ligand func-
tion as type-2 adjuvants and induce antigen-specific Th2 responses [102, 103].
Interestingly, STING ligand-induced type-2 responses were IRF3 and type-1 IFN
dependent and were completely abolished in IRF3- and type-1 IFN receptor-
deficient mice [102, 103]. Given that IRF3 and TBK1 are involved in host DNA-
dependent adjuvant activity by alum, these signaling molecules may be potent
therapeutic targets of particulate-induced allergic inflammation. Thus, host DNA
and DNA-sensing molecule and its signal transducers appear to augment allergic
inflammation through both known and unknown mechanisms.
74 E. Kuroda et al.
It is not only DNA that is released from damaged cells; uric acid and its crystals
are also released. We also found that higher levels of PGE2 were released during cell
death (unpublished data). Taken together, it is evident that many different types of
DAMPs released from dying cells induced by particulates promote immune
responses and exacerbate allergic inflammation.
5.5 Th2 Cytokine-Producing Cells
Particulate adjuvants including particle pollutants promote antigen (allergen)-spe-

cific IgE, and in general Th2 cytokines IL-4 or IL-13 and signal transducer and
activator of transcription 6 (STAT6) are required for class switching to IgE in B
cells. In fact, Brewer et al. demonstrated that serum IgE production was abolished
in IL-4 receptor- and STAT6-deficient mice immunized with antigen + alum [104,
105]. However, the question of how alum induces IL-4-producing cells has remained
poorly studied. Several reports observed IL-4-producing cells after the administra-
tion of alum. Jordan et al. showed that Gr-1+ IL-4-producing cells were recruited to
the injection site of alum, and Wang et al. confirmed that the alum-elicited IL-4-
producing cells were eosinophils and basophils [106, 107]. Furthermore, chitin par-
ticles, which are potent allergic inflammation inducers, also promote the recruitment
of IL-4-producing cells that are identified as eosinophils and basophils [17].
However, it has been reported that IgE responses are normal in both eosinophil-
deficient and basophil-deficient mice that are immunized with OVA + alum [69,
108]. A recent study has demonstrated that type-2, but not type-1, natural killer T
(NKT) cells appear to be required for alum-induced antibody responses by the regu-
lation of IL-4-producing T cells [109].
Recently, unique cells have been reported to play a role in the regulation of IgE
production and Th2 differentiation, namely, group 2 innate lymphoid cells (ILC2)
[110, 111]. ILC2 cells were identified by three different research groups as natural
helper cells, nuocytes, or innate helper 2 cells [112114]. ILC2 cells produce
higher amounts of IL-5 and IL-13 in response to IL-25 and IL-33 and play an impor-
tant role in protective immunity against helminth infection. In addition, recent stud-
ies have demonstrated that ILC2 cells also regulate Th2 differentiation using a
protein allergen-induced lung inflammation model [115, 116]. So far, there is no
clear evidence whether or not particulate adjuvants stimulate ILC2 cells, but this
question may be solved in the near future. Another unique cell type is follicular
helper T (Tfh) cells [117]. Tfh cells were identified as a subset of germinal center
(GC) CD4+ T cells that differ from conventional Th2 cells [118121]. Tfh cells are
reported to produce IL-4 in GC and are considered a critical source of IL-4 for IgE
production [122]. In fact, recent studies have shown that mice that are specifically
deficient in IL-4 production by Tfh cells, but not Th2 cells, displayed a significant
reduction in IgG1 and IgE after the immunization with antigen + alum; however
differentiation into Th2 cells and IL-4 production was normal in these deficient
mice [123, 124]. These results might imply that particulate adjuvants preferentially
activate Tfh cells to produce IL-4, in a cell death-dependent manner.
PM2.5 Alum PGLA

Particulates ASD Silica MSU (no allergic responses)
Microbial
Components
(LPS etc)
Cell death inflammasome
TLR2/4
???
Host DNA Syk
MyD88 IL-1
IRF3
IL-18
TBK1
(IL-1 ?)
??? PGE2
DC activation synthesis
IL-4/IL-13 producing cells, ILC2??

STAT6
Th2 cells, eosinophils
Follicular Helper T cells

Germinal Center
IgG1/IgE-priducing B cells, mast cells
Fig. 5.2 Summary of proposed models of the mechanism of action of particulate adjuvant.
Particulate adjuvants induce immune responses through TLR signaling, inflammasome activation,
or DAMP release from damaged cells
5.6 Conclusion
We summarized the mechanism of action of particulate causing allergy in the

immune system in Fig. 5.2. In this review, we focused on the effect of particulate
adjuvants on allergic inflammation. To date, many studies have demonstrated that
some kind of particulates induce and exacerbate allergic inflammation as character-
ized by the activation of eosinophils and the induction of serum IgE. However, the
mechanistic details remain to be determined, as does the mode of action of alum
despite the fact that alum has been extensively employed as a human vaccine adju-
vant. Advances in particulate adjuvant research could open new possibilities for the
treatment of particulate-induced allergic disorders.
Declaration of Interest EK received a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan (grant number 24591145
and 15K15390) and Takeda Science Foundation and the Mochida Memorial Foundation for
Medical and Pharmaceutical Research. KJI and CC were supported by a Health and Labor Science
Research Grant Adjuvant Database Project from the Japan Agency for Medical Research and
Development (AMED).
76 E. Kuroda et al.
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Chapter 6
Traditional and Emerging Occupational
Asthma in Japan
Kunio Dobashi
Abstract Occupational asthma (OA) is one of the most common forms of occupa-
tional lung disease in many industrialized countries, and it accounts for 915 % of
adult asthma. If a worker with an occupational allergic disease doesnt consider it
an occupational disease, or if affected workers bear it and take no measures or treat-
ment, extensive exposure at the workplace will persist. These cause the disease to
worsen or become refractory. Sometimes, patients might lose their job and face
economic difficulties. Therefore, we should always take the possibility of OA into
consideration and obtain a detailed history from patients. When OA is diagnosed,
patients should avoid allergen exposure, and the workplace environment should be
improved, as well as adequate drug therapy being provided. This paper covers the
history, current state, and the published first guideline for diagnosis and manage-
ment of occupational allergic diseases in Japan.
Keywords Occupational asthma Konjac asthma Guideline Japan
6.1 History of OA in Japan
Occupational asthma (OA) is one of the most common forms of occupational

lung disease in many industrialized countries, and it accounts for 915 % of adult
asthma [1].
The first case of OA in Japan was reported in 1926 as Asthma attack induced by
working with American red cedar wood. When large amounts of American red
cedar were imported for reconstruction after the Great Kanto Earthquake, carpen-
ters suffered from asthma when processing this cedar. An epidemiological study
revealed that approximately 10 % of carpenters were suffering from OA due to
cedar. Therefore, import of the cedar was stopped and such OA was no longer seen.
K. Dobashi (*)
Graduate School of Health Sciences, Gunma University,
3-39-22 Showa-machi, Maebashi, Gunma 371-8514, Japan
e-mail: dobashik@gunma-u.ac.jp

DOI 10.1007/978-981-10-0351-6_6
84 K. Dobashi
After IgE was discovered in 1966, OA began to be studied from an immunologi-

cal perspective. The first OA to be identified in Japan was konjac asthma. At that
time, bronchial asthma apparently caused by Maiko powder was known among
the residents living near konjac milling plants and the employees of these plants. A
group from the First Department of Internal Medicine at Gunma University studied
a detailed field survey in Shimonita that identified OA induced by inhaling Maiko
powder, which they called konjac asthma and reported in 1951 [2]. After that, many
OAs were reported as sea squirt asthma, silkworm phosphorus hair asthma, buck-
wheat asthma, silkworm cocoon asthma, and shiitake mushroom asthma in 1996,
1996, 1970, 1971, and 1985, respectively [3, 4].
6.2 Epidemiology of Occupational Asthma in Japan
The rate of the OAs is about 215 % of all asthmatics in Japan. However, we do not
have correct data of incidence and prevalence of OA. Since it is possible that many
patients are treated for asthma without diagnosis of OA, the actual prevalence is
probably higher.
Among persons involved in specific types of work, the prevalence of OA depends
on the allergens to which they are exposed or the work environment, and there are
many reports about its prevalence (Table 6.1) [5, 6].
It is a serious problem that a high prevalence was shown around some factories
because allergens released from the workplace cause residents living around the
plants to develop asthma. We found that many residents living around a konjac fac-
tory had konjac asthma, and we proposed that this should be called environmental
asthma (Table 6.2) [7].
Table 6.1 Estimated Type of work Prevalence (%)

preverence of work-related
Konjac maker 5.0
asthma from cross-sectional
studies in Japan Sericulturist 9.0
Polyurethane industry worker 16.4
Worker in plastic greenhouses 4.6
of strawberry
Worker in plastic greenhouses 5.0
of shiitake mushroom
Table 6.2 The number of Konjac asthma patients depend on the distance from the factories
Distance from The number of Konjac asthma The number of non-Konjac asthma
factories patients patients
Within 300 m 46 17
3001000 m 4 13
More than 1000 m 1 20
6 Traditional and Emerging Occupational Asthma in Japan 85
6.3 Allergens
The causative allergens are varied and sometimes unexpected. A part of substances
that have been reported in Japan are summarized in Table 6.3 [7].
Causative allergens are divided into allergens of high molecular weight, such as
those derived from animals and plants, and allergens of low molecular weight, such
as chemicals and metals. According to the development of industry, the incidence of
asthma induced by high molecular weight compounds is decreasing. On the other
hand, asthma induced by low molecular weight substances is increasing and has
become a serious problem, recently [5, 6]. The problems in OAs caused by chemicals
are that the specific IgE antibody cannot be easily detected and diagnosis is difficult.
Table 6.3 Reported occupational allergens in Japan

Allergen Occupation Year and author
Plants Cereal Amorphophallus Konjac maker 1951 Shichijo
konjac
Buckwheat Buckwheat miller. soba 1971 Nakamura
restaurant worker
Wheat flour Baker. confectionary makers 1971 Jyo
Barley flour Miller 1991 Noda
Wood Western red cedar Wood processing industry 1926 Seki
particle worker
Zelkova Wood processing industry 1982 Katsuya
worker
Mulberry Furniture making industry 1969 Nakamura
worker
White birch Wood processing worker 1979 Takamoto
Lauan Wood processing worker 1968 Aoki
Quince Wood processing worker 1975 Takahashi
Boxwood Wood processing worker 1985 Tawara
Others Coffee beans Manufacturing plant worker 1985 Shirakawa
powder
Sesame Manufacturing plant worker 1990 Tadokoro
sesame oil
Tea leaf
Fresh top Tea picking worker 1976 Ebihara
Component Tea manufacture worker 1989 Otsuka
of tea leaf
Tomato (component Worker in plastic 1980 Saito
in stalk) greenhouses
Lettuce (component Worker in plastic 1980 Saito
in stalk) greenhouses
Fuzz of melon Worker in plastic 1980 Masuyama
greenhouses
(continued)
86 K. Dobashi

Allergen Occupation Year and author
Animal Silk Silk textile industry worker 1966 Nakamura
Sea squirt Oyster farm worker 1964 Jyo
Alcyonarian Japanese spiny lobster 1989 Onizuka
Animal hair Writing brush maker 1968 Kikuchi
Mixed fertilizer Fertilizer factory worker 1982 Usami,
(fish, crab) 1991 Kasiwagi
Sardine powder Dried sardine factory worker 1987 Takamoto
Coat of rat and Researcher 1972 Kobayashi
guinea pig
Pollen, Pollen Sugar beet Researcher of sugar beet 1970 Matsuyama
spore Rose Researcher of rose 1978 Saito
Orchard grass Commercial grower of 1971 Nakazawa
orchard grass
Strawberry Worker in plastic 1973 Kobayashi
greenhouses of strawberry
Peach Commercial grower of peach 1973 shida
Apple Artificial pollination worker 1978 Sawada
Grape Worker in plastic 1984 Tsukioka
greenhouses
Pepper Worker in plastic 1985 Okumura
greenhouses
Spore Shiitake mushroom Worker in plastic 1968 Kondo
greenhouses
Club moss Dental technician 1969 Nakamura
Smut fungus Commercial grower of 1983 Asai
wheat.
Metal, Drug Diastase Pharmacist; at drugstore; 1970 Fueki
chemical Pancreatin Pharmacist; at drugstore; 1971 Nakamura
Semisynthetic Pharmacist; at drugstore; 1974 Kanetani
penicillin
Metal Dichromate Workers of cement 1972 Fueki
producing industry
Chloroplatinate Industry worker 1984 Shima
Chemical TDI MDI Polyurethane industry 1970 Shima
worker, painter
Ethylenediamine Plastic processing worker 1979 Nakazawa
Tetryl (explosive) Pyrotechnist 1989 Inagaki
Acrylic resin Painter 1990 Nakamura
emulsion
6.4 Traditional Occupational Asthma in Japan
6.4.1 Konjac Asthma
As I have described previously, the first OA to be identified immunologically in

Japan was konjac asthma [2]. Konjac root is dried and ground into powder in the
process of manufacturing the food known as konjac (no calorie food). Maiko is a
fine konjac root powder that is blown by air pressure to obtain konjac powder for
commercial use. Much of the Maiko powder is dispersed in the air and induces
asthma in the plant workers by inhalation. The prevalence of konjac asthma was
16.6 % among employees in konjac mills, and the age of onset was different but
mostly under 30 years.
The purified allergen of konjac asthma named Ag40D-2 is an acidic protein of
about 24,000 daltons. Its ratio of basic to acidic amino acids is 1:3.7 and it induces
a strong P-K reaction.
The immediate skin reaction to a purified Maiko powder allergen is positive in
100 % of konjac asthma patients, but negative in non-konjac asthma patients.
When the asthma was discovered, konjac making was an important industry in
the Shimonita area and 40 % of the population were involved in producing konjac
flour. Therefore, specific immunotherapy was developed because of the difficulty in
changing jobs. When its efficacy was assessed, it was remarkably effective in 6/35
persons (17.1 %) and was effective in 18 (51.4 %) [3, 4].
After the konjac asthma was reported, companies began to improve the work
environment. As a result of great effort, no one has developed konjac asthma since
the late 1980s.
6.4.2 Sea Squirt Asthma
Sea squirt asthma is triggered by the inhalation of fluid from protochordate sea
squirts that is adherent to cultured oysters. Cultivation of oysters in the Hiroshima
region has been done for 400 years, and many people are engaged in the task of
oyster husking. There were no reports before World War II, but employees com-
plained of the onset of asthma associated with their work from around 1960. This
asthma was reported in 1963 by Mitsui. In addition, detailed studies revealed that
this type of asthma was induced by the inhalation of sea squirt components adherent
to oysters. Such OA was named sea squirt asthma in 1966 [8].
The cause of its onset was improved farming methods that allowed farming of
oysters in deep water since around 1952, so that sea squirts became attached to the
oysters. Because work was often done under rough conditions with poor ventilation,
workers inhaled a lot of sea squirt components.
From the investigation done at the time, the prevalence was 29 % (443 out of
1,528 people) and it reached 45.8 % in some towns. Because the industry has mostly
88 K. Dobashi
female employees, there is a majority of female patients. Half of the patients develop
asthma within 5 years of starting work.
Separation and purification of sea squirt allergen was carried out and four aller-
gens (H, Gi-rep, Ei-M, and DIIIa) were identified. Especially Gi-rep and Ei-M were
effective for immunotherapy.
The skin reaction to sea squirt allergen is positive in 91.3 % of sea squirt asthma
patients. When an allergen inhalation challenge test was done with sea squirt aller-
gen, four out of nine sea squirt asthma patients were positive.
Initially immunotherapy was done with the crude allergen and the efficacy rate
was high at about 75 %. However, immunotherapy with the crude allergen caused
side effects such as induction of asthma or urticaria. In contrast, therapy with
the purified allergen has a higher efficacy rate of 91.5 % and causes fewer side
effects [8].
As a result of great effort to improve work environments, the number of patients
has recently shown a significant decrease due to improvement of the work
environment.
6.5 Emerging Occupational Asthma
6.5.1 High Molecular Weight Allergen
The pollen of vegetables and fruits or spores of mushrooms have become causative
allergens along with the increase of greenhouse culture. Especially, shiitake mush-
room, tomato, and strawberry were not recognized as causing asthma when open-
field cultivation was common.
Furthermore, it was reported that a furniture craftsman developed asthma by
inhalation of the dust of Albizia falcataria (Falcata wood), which is a broad-leafed
tree and began to be imported recently.
6.5.2 Low Molecular Allergen
There have been reports about occupational allergy induced by ortho-phthalaldehyde,

which is used as a disinfectant solution for fiberscopes. Cases of ortho-
phthalaldehyde-induced anaphylaxis began to be reported from around 2006. For
example, anaphylaxis has occurred immediately after observation by a laryngeal
fiberscope. Since various new chemicals will be developed in the future, we always
have to pay attention to allergies caused by chemicals.
6.6 The First Guidelines for Occupational Allergic Diseases

in Japan
6.6.1 Necessity of Guideline
It is extremely important to identify occupational allergic disease cases in their early

stages and take appropriate preventive measures for the social lives of patients. A
guideline was released in Canada as long ago as 1998, while the American Thoracic
Society (ATS) guidelines were published in 2005 [1]. In the same year, other guide-
lines were published in the United Kingdom. Other guidelines that described diag-
nosis and management in detail were published by The American College of Chest
Physicians, while more guidelines were released in Spain (in 2006) and in Singapore
(in 2008). These guidelines show wide recognition of the importance of occupa-
tional asthma.
In Japan, a large number of case reports have been accumulated on occupational
allergic diseases. However, because of the occupational features of the diseases,
only case reports have been presented in many cases. Although guidelines for indi-
vidual allergic diseases have been published by allergologic associations, the
descriptions of occupational factors are generally minimal. It is extremely important
that the guideline for diagnosis and management of occupational allergic diseases
have been published in 2013 for the first time in Japan [5] (Fig. 6.1).
This guideline is designed to assist healthcare professionals engaging in ordinary
diagnosis and management of allergic diseases to practice early detection and treat-
ment and early prevention in patients with allergic diseases induced and worsened
by occupational factors. We hope that this guideline will be used for ordinary diag-
nosis and management of occupational allergic diseases and help the patients.
6.6.2 The Structure of the Guideline
The guideline has a basic structure in which clinical questions are set with reference
to Medical Information Network Distribution Service (MINDS); statements by the
committee are listed; recommendation grades and evidence levels are defined;
descriptions and references are indicated. Also, legal aspects are written in full.
As for occupational allergic diseases, because new substances have been con-
tinually produced due to technical innovation and working environments have been
changing due to changes in industrial structures, new OAs can always arise. We
have revised the guideline in 2016 and will continue to revise it every 3 years, in
order to maintein a high level of evidence for the guideline.
90 K. Dobashi
Fig. 6.1 First Japanese guideline for occupational allergic diseases 2014
6.7 Problems Related to Occupational Asthma in Japan
1. Due to advances in medication, achieving control of symptoms medically tends

to be emphasized, and the search for causative allergens tends to be neglected.
Thus, physicians often do not try to identify the causative allergen.
2. Poor surveillance data.
3. Poor regulation by law.
4. The work environment has improved in large enterprises under the direction of
the government, but the smaller companies are not considered to have made
enough effort in some cases.
6.8 Action Plans
1. Make surveillance system/checkup lung conditions of workers regularly.

2. Revise the guidelines of prevention and control of OA at stated periods.
3. OA information center and homepage are required.
4. Share information on OA in other countries.
5. Education about OA for workers, employers, healthcare providers, and govern-
ment agencies.
References
1. Mapp CE, et al. Occupational asthma. Am J Respir Crit Care Med. 2005;172(3):280305.
2. Shitijyo K. The study of Konnyaku asthma. Kitakannto Med J. 1951;1:2930.
3. allergy, J.c.o.o, editor. Occupational asthma. Tokyo: Asakura Publishing Co., LTD; 1973.
4. allergy, J.c.o.o, editor. Occupational allergy. Tokyo: Buneido Publishing Co., LTD; 1983.
5. Committee for Japanese Guideline for Diagnosis and Management of Occupational Allergic
Diseases. Japanese guideline for diagnosis and management of occupational allergic diseases
2013. Tokyo: Kyowa Kikaku; 2013 (in Japanese).
6. Dobashi K, et al. Japanese guideline for occupational allergic diseases 2014. Allergol Int.
2014;63(3):42142.
7. Dobashi K. Occupational allergy. In: Fukuda T, editor. Sogo allergy-gaku. Tokyo: Nanzando;
2010.
8. Katsuya T. Hoya (Sea-Squirt) asthma (Japanese). Occup Environ Allergy. 2005;12:115.
Chapter 7
Skin Sensitization Model Based on Only
Animal Data by Qualitative Structure-Toxicity
Relationships (QSTR) Approach
Kazuhiro Sato, Kohtaro Yuta, and Yukinori Kusaka
Abstract Contact dermatitis is by far the most common form of occupational skin
illness. In silico assessment of skin sensitization is increasingly needed owing to the
problems concerning animal welfare, as well as excessive time consumed and cost
involved in the development and testing of new chemicals.
We previously made skin sensitization model from human and animal data and
reported. Its accuracy was 61.2 % (sensitivity 60.7 %, specificity 62.8 %) by external
validation. This time we made skin sensitization QSTR model from only animal
data (local lymph node assay (LLNA), 471 chemicals) by using K-step Yard sam-
pling (KY) methods (US Patent No. 7725413, 2010) and 1 model KY method (US
Patent Application).
A total of 320 compounds (212 positive sensitizers and 108 negative sensitizers)
were used in this study. Two hundred and eighty-eight compounds were used to
make a QSTR model and external validation study was performed by 32 com-
pounds. The concordance of QSTR prediction for LLNA data was 71.9 % (sensitiv-
ity 54.5 %, specificity 81 %) and better than previous report. The concordance was
better than previous time and indicates that the data of human and animal study
were qualitatively different from each other.
Keywords Skin sensitization Alternative methods Qualitative structure-toxicity

relationships (QSTRs) Animal welfare
K. Sato (*) Y. Kusaka

Department of Environmental Health, School of Medicine, University of Fukui,
Fukui 910-1193, Japan
e-mail: satokazu@u-fukui.ac.jp
K. Yuta
In Silico Data Co Ltd, Narashino, Chiba 275-0025, Japan

DOI 10.1007/978-981-10-0351-6_7
94 K. Sato et al.
7.1 Introduction
In occupational health, occupational skin disorders are the most common. Among
them, contact dermatitis is by far the most common form of occupational illness [1].
Under the new European Union (EU) Registration, Evaluation, and Authorization
of Chemicals (REACH) rules, all chemicals in the EU that are produced or imported
in quantities of more than 1 ton per annum need to be assessed as potential human
and environmental hazards, for example, in terms of their carcinogenicity and
human sensitivity, such chemicals also need to be determined. REACH calls for
increased use of hazard assessment alternatives such as in vitro methods and
QSTRs [2].
Since only two in vitro replacements are currently available for skin sensitization
[3, 4], the use of QSTR approaches presents an attractive alternative [5]. One of the
most difficult subjects in QSTR research is computer classification and prediction
of chemical toxicity of compounds. This is because (1) there is large structural
diversity among samples, (2) the sample number is enormously large, and (3) high
classification and prediction rates are required. Nonlinear discriminant functions,
such as neural network (NN), support vector machine (SVM), and AdaBoost, some-
times provide higher classification rate than that of linear methods. However, non-
linear methods are often accompanied by over-fitting which lowers prediction rate
significantly.
Previously, we made the QSTR models for skin sensitization [6, 7], which are
statistically based model [6], and the model by KY methods [7, 8] using animal-
human mixed data based on human epidemiological studies, case reports, or vali-
dated animal studies (guinea pig maximization test, Buehler guinea pig test [9, 10].
The correct classification of our previous model by KY methods was 61.2 % (sensi-
tivity 60.7 %, specificity 62.8 %). These KY methods can be applied to a linear and
nonlinear discriminant function. The KY methods could classify a set of Ames test
samples (6,965 compounds, 2,932 positive, 4033 negative) into two classes (posi-
tive/negative) correctly by 23 steps (data are not shown).
In this paper, we illustrate the KY methods, 1 model KY methods (US Patent
Application), and only animal data (murine local lymph node assay) [11] based on
our new model by KY methods and 1 model KY methods [7, 8].
7.2 Materials and Methods
7.2.1 Chemicals
Positive and negative 471 chemicals were obtained from the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Test
Method Evaluation Report, the reduced murine local lymph node assay [11]. The
criteria of positive data are more than three of stimulation index. However,
7 Skin Sensitization Model Based on Only Animal Data by Qualitative 95
inorganic chemicals, organic metal compounds, and polymers are special com-
pounds and cannot be analyzed with general organic compounds in computational
chemistry. Therefore, we deleted these chemicals and finally assessed 320 (212
positive and 108 negative) compounds. Two hundred and eighty-eight chemicals
were used for learning set and 32 chemicals are used for external validation study
(10 % cross-validation).
7.2.2 Parameters and Discriminant Functions
A total of 320 compounds (212 positive and 108 negative) were used for analysis.
Two hundred and eighty-eight chemicals were used for learning set and 32 chemi-
cals were used for 10 % cross-validation study (10 % CV). Parameters were gener-
ated from 2-D and 3-D structures of the compounds. The generated parameters were
reduced by various feature selection (e.g., removing low appearance parameter,
high correlation, or multicollinearity) methods. The KY methods were applied [7,
8]. In this case, AdaBoost and the iterative least squares linear discriminant func-
tions (TILSQ) were used for generating discriminant functions.
7.2.3 KY Methods
Existing binary classifiers generate only one discriminant function in order to clas-
sify a sample set into two classes (Fig. 7.1). The same is true in the case of newly
developed nonlinear classification methods, such as NN, SVM, and AdaBoost.
Fig. 7.1 Linear discriminant function to classify all samples [7]

96 K. Sato et al.
These nonlinear methods sometimes provide a higher classification rate than those
of linear methods. However, if the samples highly overlapped one another, linear
and nonlinear methods cannot classify the samples correctly into two classes
(Fig. 7.2).
In the process of the KY methods, two different types of discriminant functions
were created to determine positive, negative, and gray zones (Fig. 7.3). One of the
discriminant functions is called as all-negative (AN) model and the other as all-
positive (AP) model. The AN model classified AN samples in the sample set cor-
rectly and the AP model classified all-positive samples correctly. The samples which
were classified as negative samples by AN model and positive samples by AP model
belonged to the gray zone (Fig. 7.3).
Fig. 7.2 Highly overlapped samples could not be classified completely by linear and nonlinear
discriminant function [7]
Fig. 7.3 Classification results by AP and AN sample discriminant functions. Samples in positive
zone and negative zone had high reliability of classification. Gray zone was not classified [7]
The KY methods focused on both sides of a sample space and found that there
were special spaces, which included only correctly classified samples. These two
areas have been defined as positive zone and the others as negative zone. The third
zone was named as gray zone. All samples included in the positive zone belonged
to a positive class, while all samples included in the negative zone belonged to a
negative class. On the other hand, the samples included in the gray zone could not
be determined whether they belonged to a positive or negative class since they were
highly overlapped (Fig. 7.3).
If the gray zone (1) was determined by AN1 and AP1 discriminant functions, the
gray zone (1) could be extracted and reclassified by AN2 and AP2 models to build a
new sample set. If a new gray zone (2) was determined with respect to the new
sample set, a further new sample set can be built as shown in Fig. 7.4. Repeating
these steps, all samples in the original sample set can be classified correctly
(Fig. 7.5). This is the basic concept of KY methods. The AN model and the AP
model can be generated based on any conventional linear and nonlinear discriminant
function. Therefore, KY methods can be categorized as a meta-algorithm approach.
All data analyses were performed using ADMEWORKS/Model Builder soft-
ware (Fujitsu Kyushu Systems Limited, Japan).
Fig. 7.4 Improvement of classification rate by KY methods. Correctly classified positive and
negative samples are removed and the gray zone samples were reconstructed and reclassified in the
new sample space by new discriminant functions at the next step [7]
98 K. Sato et al.
Fig. 7.5 Meta-algorithm repetition of reclassification of gray zone (KY methods). High reliability
zone (correctly classified samples) was removed and gray zone was reclassified, and the sample
space was reconstructed by new discriminant functions at the next step. All samples were correctly
classified at the final step [7]
7.2.4 1 Model KY Methods
1 model KY methods are simple, easy, and delicately manipulated compared with
ordinary 2 model KY methods (Fig. 7.6).
7.3 Results
In this study, AN and AP discriminant functions at step 1 were generated by

AdaBoost. The final discriminant function was generated by TILSQ. All 288 com-
pounds were perfectly classified by two steps.
The correct classification (10 % CV) of the KY methods was 71.9 % (sensitivity
54.5 %, specificity 81 %). That of 1 model KY methods was 69 %.
Fig. 7.6 1 model KY methods (overview)
7.4 Discussion
Since the implementation of Animal Welfare Guideline 86/09/EC in 1986, it is the

declared policy of EU institutions to support the development and use of alternative
methods of testing chemicals, that is, of any method that can be used to reduce,
replace or refine the use of animal experiments in biomedical research, testing or
education [12].
However, only two in vitro replacements are currently available for skin sensiti-
zation [3, 4]. Therefore, several QSTR-related systems have been developed for
skin sensitization. These are Toxicity Prediction Komputer-Assisted Technology
(Accelrys Inc., CA, USA; TOPKAT) and Multi-Computer Automated Structure
Evaluation (MultiCASE Inc., Ohio, USA; M-CASE), which are both statistically
based; Deductive Estimation of Risk from Existing Knowledge (DEREK) for
Windows (DfW, LHASA Ltd., Leeds, UK), which is knowledge based; and Times
100 K. Sato et al.
Metabolism Stimulator for Skin Sensitization (LMC, University of Bourgas,

Bulgaria; TIMES-SS), which is a hybrid [13, 14].
In this study, the KY methods could be applied to this QSTR study. All 320 com-
pounds were classified perfectly by a total of two steps. If only linear discriminant
function is performed, these new methods always attain perfect classification rate
without over-fitting, which causes lower prediction rate. This QSTR model applied
AdaBoost and TILSQ. The correct classification rate of our previous QSTR model
for skin sensitization by KY methods [7] was 62.5 %. That of this model was 71.9 %
and better than that of previous study. These data indicated that the data of human
and animal studies might be qualitatively different from each other. Guinea pig
maximization test, Buehler test, and murine LLNA have only a 7273 % total accu-
racy of predicting actual human skin sensitizer [15, 16]. Our results might be appro-
priate. More research is needed to improve prediction rate.
We concluded that this QSTR system is thought to be applicable to initial predic-
tion of skin sensitizing ability of untested chemicals and that the KY methods were
promising tool in QSTR technology (classifying and predicting toxicity
compounds).
Acknowledgments This work was supported by JSPS KAKENHI Grant Number 25293148.
References
1. Diepgen TL, Coenraads PI. 8. Occupational contact dermatitis. In: Rustermeyer T, Elsner P,
John SM, Maibach HI, editors. Kanervas occupational dermatology, vol. 1. Heidelberg:
Springer; 2012. p. 5183.
2. EU (2006) Regulation (EC) 1907/2006 of the European Parliament and the European Council
of 18 December 2006 concerning the Registration, Evaluation, Authorization and Restriction
of Chemicals (REACH), establishing a European Chemical Agency, amending Regulation
1999/45/EC and repeating Council Regulation (EEC) No 93/793 and Communication
Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission
Directive 91/155/EEC, 93/677/EEC and Commission Directive 91/155/EEC, 93/677/EEC,
93/105/EEC and 2000/21/EEC, Off J Eur Comm L vin P eds.
3. OECD (2015) Test guideline on an In Chemico Skin Sensitization: Direct Peptide Reactivity
Assay (DPRA). OECD Guideline for the Testing of Chemicals No. 442C.
4. OECD (2015) Test guideline on an in vitro skin sensitization: ARE-Nrf2 luciferase test method.
OECD guidelines for the testing of chemicals No. 442D..
5. Patlewicz G, Aptula AO, Roberts DW, Uriarte E. A minireview of available skin sensitization.
(Q)SARs/expert systems. QSAR Comb Sci. 2008;27:6076. doi:10.1002/qsar.200710067.
6. Sato K, Umemura T, Tamura T, Kusaka Y, et al. Skin sensitization study by quantitative
structure-toxicity relationships (QSAR). AATEX. 2009;14:9409. doi:10.11232/aatex.14.940.
7. Sato K, Umemura T, Tamura T, Kusaka Y, et al. Skin sensitization study by a new qualitative
structure-toxicity relationships (QSTR) approach: K-step Yard Sampling (KY) methods. J Oral
Tissue Eng. 2012;9(3):16773. doi:10.11223/jarde.9.167doi.org/10.11223/jarde.9.167http.
8. K-step Yard sampling (KY) methods: Kohtaro Yuta, US Patent No: US7725413 B2 May 25,
2010.
9. Deutsche Forshungsgemeinshaft (DFG). IV. Sensitizing substances. In: List of MAK and BAT
values. Alley-VCH, 2008. Weinheim, pp. 15873.
10. Coz CJL, Lepoittevin JP. Dictionary of contact allergens: chemical sources, sources and refer-
ences. In: Frosch PJ, Menne T, Lepoittevin JP, editors. Contact dermatitis. 4th ed. Berlin:
Springer; 2006. p. 9431105.
11. ICCVAM Test Method Evaluation Report. The reduced murine local lymph node assay: an
alternative test method using fewer animals to assess the allergic contact dermatitis potential
of chemicals and products. NIH Publication No. 09-6439. https://ntp.niehs.nih.gov/iccvam/
docs/immunotox_docs/llna-ld/tmer.pdf. Accessed 15 Dec 2015.
12. Lilienblum W, Dekant W, Foth H, Gebel T, et al. Alternative methods to safety studies in
experimental animals: role in the risk assessment of chemicals under the new European
Chemicals Legislation (REACH). Arch Tocicol. 2008;82:21136. doi:10.1007/
s00204-008-0279-9.
13. Patlewicz G, Aptula AO, Uriarte E, Roberts DW, et al. An evaluation of selected global (Q)
SARs/expert systems for the prediction of skin sensitization potential. SAR QSAR Environ
Res. 2007;18:51541. doi:10.1080/10629360701427872.
14. Patlewicz G, Dimitrov SD, Low LK, Kern PS, et al. TIMES-SS--a promising tool for the
assessment of skin sensitization hazard. A characterization with respect to the OECD valida-
tion principles for (Q)SARs and an external evaluation for predictivity. Regul Toxicol
Pharmacol. 2007;48:22539. doi:10.1016/j.yrtph.2007.03.003.
15. Haneke KE, Tice RR, Carson BL, Margoin BH, Stokes WS. ICCVAM evaluation of the murine
local lymph node assay. Data analyses completed by the National Toxicology Program
Interagency Center for the Evaluation of Alternative Toxicological Methods. Regul Toxicol
Pharmacol. 2001;34:27486. doi:10.1006/rtph.2001.1498.
16. The murine local lymph node assay. The results of an independent peer review evaluation
coordinated by the interagency coordinating committee on the validation of alternative meth-
ods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative
Toxicological Methods (NICEATM). NIH publications no. 99-4494, 1999. Research Triangle
Research.
Chapter 8
Nonindustrial Indoor Environments
and WorkRelated Asthma
Nicola Murgia, Ilenia Folletti, Giulia Paolocci, Marco dellOmo,

and Giacomo Muzi
Abstract Work-related asthma is one of the most relevant work-related diseases

worldwide, causing a high socio-economical burden. In the last decades, many
countries experienced huge modifications in work organisations. These changes
made people to move from traditional sectors to the tertiary sectors and non-
industrial indoor working environments. Non-industrial indoor workplaces are
characterised by a new concept of building, with a new structure, new materials,
forced ventilation, tight construction and a potential exposure to new risk factors for
work-related asthma, such as new chemicals and biological agents able to cause or
exacerbate asthma. The actual scientific evidence suggests an increased risk of
asthma among workers exposed to cleaning agents in indoor working environment
and moulds in damp buildings. Also volatile organic compounds (VOCs) and envi-
ronmental tobacco smoke could be considered triggers of asthma, even if their role
is still under debate. Because of the increasing numbers of subjects working in non-
industrial indoor environments and the scientific evidence of an increased risk of
asthma in indoor environment, there is a need of public health intervention towards
the prevention of work-related asthma, also in this specific setting.
Keywords Asthma Indoor air Moulds Cleaning Dampness
8.1 Introduction
Work-related asthma is one of the leading work-related diseases worldwide; 15 % of

all cases of asthma are related to workplace exposures [1]. Current guidelines and
statements divide work-related asthma in occupational asthma, directly caused by a
specific exposure at the workplace, and work-exacerbated asthma, a form of com-
mon asthma which is aggravated by occupational exposure [2]. Many occupational
N. Murgia (*) I. Folletti G. Paolocci M. dellOmo G. Muzi

Section of Occupational Medicine, Respiratory Diseases and Toxicology, University of
Perugia, Piazzale Severi 1, 06100 Perugia, Italy
e-mail: nicola.murgia@unipg.it

DOI 10.1007/978-981-10-0351-6_8
104 N. Murgia et al.
exposures, around 250, have been found to cause directly or to exacerbate asthma.
Chemicals causing or exacerbating asthma are usually divided into high molecular
weight (HMW) agents (e.g. flour, animal allergens, latex, etc.), low molecular
weight (LMW) agents (e.g. isocyanates, acrylates, etc.) and irritants. HMW and
LMW show often an immunologic mechanism of action which is IgE mediated
(HMW) or through a non-IgE Th2 response (LMW), while irritants are not showing
a specific immunologic pattern, but sometimes an unspecific activation of the
humoral response [2, 3].
These exposures may occur in many workplaces, both industrial and non-
industrial. One important working environment, where exposure to asthmogenic
substances may happen, is the non-industrial indoor workplaces.
8.2 Non-industrial Indoor Environment
The non-industrial indoor setting is a composite life and work environment used for
housing (private houses, hotels, etc.), common social and health structures (hospi-
tals, schools, etc.), leisure activities (bars, cinemas, sport halls), transportation
(buses, trains, boats, etc.) and work (offices, hospitals, etc.). Nowadays, in North
America, Europe and Japan, people are spending 8090 % of their life in an indoor
environment [4] and in their houses but also at work.
Modern indoor workplaces are very different from the past for their external and
internal structure. They are usually placed in suburban areas and constituted by an
internal load-bearing core and an external light frame. This structure allows to reach
a considerable height, which is useful to reduce the land cost. Anyway, this kind of
structure needs some adjustment to guarantee comfort and to reduce costs related to
heating and cooling of the indoor environment. In new non-industrial indoor envi-
ronments, measures essential to ensure comfort and reduce the costs are:
Massive use of insulating material, which was asbestos in the past decades and
now is man-made mineral or ceramic fibres
Artificial ventilation, usually controlled remotely, which implies also the pres-
ence of tight buildings, to assure control and reduce the dispersion of heating
or cooling
Artificial lighting
Besides, in modern non-industrial indoor environments, for design and economi-
cal reason, interiors have been changed a lot during the last decades. Furnitures are
now made mainly with light, low-cost, composite materials and resins, such as ply-
wood, chipboard and urea-formaldehyde resins. Floors and walls are covered by
plastic carpets. Computers and printers are the main working tools encountered in
these workplaces. Finally, to ensure cleanness and hygiene, a wide range of chemical
cleaning agents are used, particularly in indoor spaces dedicated to special purposes
(e.g. hospitals and schools).
8 Nonindustrial Indoor Environments and WorkRelated Asthma 105
This indoor structure has also a great impact on work organisation and, despite
obvious differences between countries in terms of materials, architecture and needs,
is revolutionising the concept of work as human beings have thoughts in the previ-
ous centuries [5].
8.3 Indoor Air Pollutants
The new way of projecting and constructing new living and working settings, with
the use of many new chemicals to build and keep clean the structure, has influenced
greatly the indoor air quality. The American Society of Heating, Refrigerating and
Air-conditioning Engineers, in its 2010 standard, defines indoor air as acceptable
when known toxic chemicals are not present or below noxious concentrations and
when the majority (>80 %) of people living or working in that environment is not
reporting symptoms or diseases. Non-industrial indoor air could contain many
chemicals as VOCs, ozone, particulate matter, asbestos and man-made fibres and
environmental tobacco smoke. Moreover, indoor air could be contaminated by bio-
logical (moulds, mites, bacteria and viruses) and physical (radon) hazards [6].
The source of indoor air pollution is mainly outdoor air but also building and
furniture material release, human activity in the indoor environment, cleaning
agents and poor maintenance of heating and ventilation system.
8.3.1 Physical Pollutants
8.3.1.1 Temperature, Ventilation and Humidity
Indoor environment temperature is one of the most important parameters influenc-

ing workers health and productivity. High temperature and low humidity have been
associated with respiratory and cutaneous symptoms, such as airway, eye and skin
dryness, but also with more general symptoms (headache). As a matter of fact, low
humidity and high ventilation have been associated with a quicker evaporation of
the lacrimal film in indoor workers [7]. On the contrary, a too high humidity seems
to increase discomfort in indoor working environments because of an increased
release of bad smell from materials contaminated by moulds. In fact high humidity
could increase the growth of fungi and dust mites, which could affect the health and
wellness of workers staying indoor [8]. Nevertheless, temperature and humidity
could influence also the concentration of chemicals in indoor air, especially particu-
late matter and chemicals released by furniture, insulation material and carpets.
This would make difficult to understand whether symptoms related to indoor air are
associated more with physical pollutants or with the influence of physical parame-
ters on chemical composition of indoor air.
8.3.1.2 Radon
Radon is a natural radioactive carcinogenic compound, which is usually found in

indoor air. It derives from the natural release from the soil where the building is
placed. Radon high-releasing soils are volcanic rocks and materials (tuff, pozzolan,
etc.) and granites. Radon is usually concentrated in lower floors, especially those
underground, and its contamination is fostered by the natural flow between outside
cold air and inside warmer air (the so-called chimney effect); ventilation is also an
important factor which usually reduces the concentration of radon in indoor tight
environments. Radon is a known carcinogenic compound for the International
Agency for the Research on Cancer (IARC), and many studies have issued the prob-
lem of indoor air contamination in occupational and nonoccupational settings [9].
8.3.2 Chemical Pollutants
8.3.2.1 Formaldehyde
Formaldehyde is found in building materials and furniture (panels, plywood, chip-

board, medium-density fibreboard (MDF)), glues, insulating foams, resins and envi-
ronmental tobacco smoke. Formaldehyde in indoor air could come also from the
reaction between ozone and other VOCs or aliphatic hydrocarbon (from printers) or
alkanes (from cleaning products and air fresheners) [10]. The amount of formalde-
hyde released in the environments is higher for newly constructed living and work-
ing environments, including newly produced furniture. Moreover, formaldehyde
concentration is influenced by temperature and humidity of indoor air. In offices
formaldehyde concentration is around 0.045 mg/m3; for these concentrations some
worker could start to perceive formaldehyde smell as well to report various symp-
toms such as nose, throat, eyes and skin irritation, headache, sleepiness and dizzi-
ness [11]. Some studies highlighted a possible role of formaldehyde in causing
asthma and rhinitis, by the interaction with other chemicals (ozone, VOCs) and
directly enhancing the expression of adhesion molecules on nasal mucosa [12].The
IARC has classified formaldehyde carcinogenic for human beings, because of its
role in causing nasal cancer and other malignancies [13].
8.3.2.2 VOCs
VOCs are organic compounds found in indoor air, with a boiling point between 50
and 250 C. They are rather many comprise of aliphatic and aromatic hydrocarbons,
cycloalkanes, aldehydes, terpenes, alcohols, esters and ketones [14]. Major sources
of VOCs are building materials, paints, glues, furnitures, cleaning products, printers
and photocopiers. VOCs are also produced by chemical reaction between other
chemicals, especially ozone and nitrous compounds, and by moulds (the so-called
MVOCs), being responsible of the bad smell often perceived in indoor environ-
ments. Exposure to VOCs has been associated with airway irritation, inflammation
and obstruction and general symptoms (sleepiness, dizziness), even if this finding
has not been always confirmed [15].
8.3.2.3 Ozone
Ozone concentration in indoor air is related to ozone in outdoor air, to ventilation,

to reaction with other chemicals (ozone-initiated indoor chemistry) and to the
release from furniture and building materials. Chemicals usually reacting with
ozone in indoor environments are isoprene, styrene, terpenes, squalene and satu-
rated and unsaturated fatty acids from furniture, carpets, cleaning products and air
fresheners. One important source of ozone pollution in indoor air is the release by
laser printers, photocopiers, electrostatic filters and precipitators [16].
Exposure to mild concentration of ozone (160 g/m3) can cause airway irrita-
tion, inflammation, obstruction and bronchial hyperresponsiveness. Respiratory
symptoms associated with ozone exposure are more frequent in subjects with pre-
existent respiratory diseases. In allergic patients ozone could prime the airways
enhancing the response to inhaled allergens. Genetic could explain the large variety
of short-term reactions to ozone in different subjects. An increase in outdoor ozone
concentration has been associated with an increased mortality and hospital admis-
sion for respiratory diseases. It has been also shown that outdoor exposure to ozone
is correlated with higher morbidity for cardiovascular diseases [17].
8.3.2.4 Phthalates
Phthalates are a large family of chemical compounds used in the indoor environ-
ment to increase the flexibility and elasticity of polyvinylchloride-based carpets.
Even if phthalates are mainly introduced in the body by the oral route, respiratory
exposure by indoor air has been demonstrated. Phthalates are mainly known as
endocrine disruptor, but some study pointed out a possible role in allergy and
asthma [18].
8.3.2.5 Particulate Matter
Particulate matter (PM) is often classified by its diameter in ultrafine (PM 0.1), with
a diameter <0.1 m; in fine (PM 2.5), with a diameter <2.5 m; and finally in PM10,
with particles <10 m. Fine particles are mainly produced during combustion and
larger particles (PM10) are usually the results of fine-particle aggregation [19].
Major sources of particulate matter in indoor air are combustion process, tobacco
smoke, laser printers and photocopiers [15]. Other less prominent sources are pol-
lens, moulds, bacteria and sprays. Usually larger particles are coming mainly from
outdoor through windows and the ventilation system. Toxic effects of particulate
matter are related to its diameter, and larger particles affect the nose, throat and
larger airways, while smaller particles, belonging to the respirable fraction, could
directly affect small airways and alveoli. Moreover, the toxicity is also mediated by
the substances (e.g. aromatic hydrocarbons, nitrous compounds, aldehydes)
absorbed onto the particles surface [20]. A large number of studies show that PM
could be a strong eye and airway irritant and could exacerbate asthma. Nevertheless,
some reports suggested a possible role of particulate matter to induce a direct alve-
oli inflammation with diffusing capacity impairment [15]. Long-term exposure to
PM is able to increase morbidity and mortality for respiratory and cardiovascular
disorders. Indoor air threshold for PM has not been established yet as it was done
for outdoor exposure to PM.
8.3.2.6 Environmental Tobacco Smoke (ETS)
Exposure to environmental tobacco smoke (also named passive smoking or

second-hand tobacco smoke) is the inhalation of tobacco smoke by non-smoking
subjects because of the presence of smokers in their same environment. ETS is one
of the most import pollutants of indoor working environment [21]. Tobacco smoke
accounts for more than 4000 toxic substances with carcinogenic, toxic and
inflammatory effects. Health effects of tobacco smoke on airways are irritation and
inflammation, causing asthma and chronic bronchitis exacerbations and increased
susceptibility to respiratory infections. Long-term exposure is correlated with air-
way impairment and chronic obstructive pulmonary disease. ETS has been classi-
fied carcinogenic by the IARC, increasing the risk of lung cancer by 1619 % in
occupational settings. Some reports suggest an increased risk also for breast cancer
[22]. Recent smoking bans in different countries have reduced a lot the exposure to
ETS. In indoor environment it has been described also the third-hand smoke
phenomenon, when after ETS cessation, smoke-related chemicals can persist in the
indoor environment absorbed onto the PM [23].
8.3.3 Biological Pollutants
In North America building dampness and the consequent mould overgrowth are
considered the principal cause of indoor-related symptoms. Mould overgrowth is
also related to ventilation and dehumidification system impairment [24]. The most
important fungal species related to indoor pollutions are Aspergillus versicolor,
Penicillium brevicompactum, Penicillium chrysogenum and Cladosporium species.
Moulds can produce a biological effect by their capacity to elicit an immune
response, causing rhinitis, asthma and even more dangerous diseases, such as
hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis. Besides,
moulds could cause a direct irritating effect on the eyes, airways and skin [25].
Dust mite is another important biological indoor pollutant capable of causing
sensitisation and consequently rhinitis, dermatitis and asthma. In the European

Community Respiratory Health Survey, indoor contamination by dust mites has
been extensively studied, finding differences in dust mites contamination across
countries, depending mainly on the indoor humidity [26].
8.4 Asthma and Non-industrial Indoor Workplaces
The relation between asthma and exposure to occupational indoor pollutants has
been extensively studied in the last decades in North America, Europe and Asia
because of the socio-economical changes, which moved people from farming and
industry to the tertiary sector.
Occupational exposures to cleaning agents, disinfectants and moulds are the
fields more frequently studied, even if also other exposures have been associated
with the onset or the exacerbation of asthma.
8.4.1 Exposure to Cleaning Agents and Disinfectant:

The Health-Care Sector
Cleaning agents are mostly irritants for the airways and thus they could act as other
factors associated with work-related irritant asthma. Glutaraldehyde, a typical dis-
infectant used in hospitals to clean endoscopes, has shown also the capacity to cause
an immunologic reaction through an IgE-mediated mechanism. Studies performed
on health-care personnel and professional cleaners working in hospital have shown
an increased risk of work-related asthma due to the exposure to traditional cleaning
agents (bleach, ammonia, detergents), especially if cleaning agents and disinfectants
are used in their sprayed form [27]. There is also a moderate scientific evidence that
exposure to specific disinfectant agents for health-care settings (glutaraldehyde,
formaldehyde, ethylene oxide) is correlated with work-related asthma and work-
related respiratory symptoms [28]. In Asia and Europe, nurses have shown an
increased risk of hospitalisation for asthma and new-onset asthma, especially if
exposed to cleaning agents [29, 30].
In hospital technician the use of ammonia or bleach as cleaning agents has been
associated with an increased risk of respiratory symptoms [31].
8.4.2 Exposure to Cleaning Agents and Disinfectant: Other

Sectors
As in the health-care sector in other sectors, cleaning agents have mainly an irritat-
ing effect. In professional cleaners the exposure to detergents seems to increase the
risk of respiratory symptoms and asthma, with an effect related also to the exposure
duration and the use of sprayed cleaning agents [32, 33]. In larger studies the
association between professional cleaning and respiratory symptoms was rather

clear, while an effect on asthma occurrence was not evident. The tasks showing a
correlation with respiratory symptoms are dewaxing, carpet and wall cleaning [34].
In other studies, exposure to ammonia was able to exacerbate a pre-existent asthma
[35]. Finally, long-term exposure to cleaning agents was associated with an acceler-
ated FEV1 decline in professional cleaners [31].
8.4.3 Exposure to Moulds
Mould exposure at work can cause allergic sensitisation and consequently a typical
allergic asthma due to fungal spores. As it is written above, exposure to Aspergillus
spp. could cause a rare form of sensitisation, the allergic bronchopulmonary asper-
gillosis, a severe disease resembling asthma in some of the first stages, but more
difficult to treat. In some case moulds can cause the production of irritating com-
pounds, such as aldehydes or particulate, which may act on the airway directly,
without a cellular immunologic response. Workplace dampness and the consequent
mould overgrowth are relevant public health problems in many countries. Office
and school dampness increases the prevalence of asthma and wheezing among,
respectively, clerks and teachers [36, 37]. Dampness was able to increase sickness
absence due to asthma in clerks [38]. Mould exposure at the workplace was also
associated with increased bronchial hyperresponsiveness [39]. Workplace damp-
ness has been found to increase the concentration of exhaled breath inflammatory
biomarkers [40]. The association between visible moulds at the workplace and
asthma is consistent also with the duration of exposure; as a matter of fact the effect
was stronger when a visible damage was present at the workplace for more than 5
years [41]. Environmental assessment of fungal growth was associated with asthma
onset [42].
8.4.4 Other Exposures
Indoor swimming pool workers have been found at higher risk of asthma because of
the exposure to disinfectants (trihalomethane, trichloramine), with a dose-response
relationship [43, 44]. In this case the effect of these substances is irritating and
usually can be resolved reducing the concentration of chemicals used to sanitise
water [45].
Workers exposed to ETS in indoor non-industrial working environments seem to
be at higher risk of asthma symptoms [46], in this case the role of passive smoking
is not completely clear, and it could act as a mixture of irritating compounds but also
enhance the sensitisation to indoor allergen, as it is happening in children exposed
to ETS. More debated is the role of occupational exposure to nitrogen oxides occur-
ring in ice sport arenas or kitchen and the occurrence of work-related asthma; in this
case the mechanism is largely unknown, even if in hockey players, a mixed neutro-
philic and eosinophilic inflammation was found analysing induced sputum, after the
training [47, 48]. Less evidence is provided about the role of ozone and particulate
released by laser printers and photocopiers on asthma onset [49, 50].
8.5 Conclusions
Economy, technology and work organisation have changed workplaces enormously

in the last 30 years. Besides, the number of workers involved in the tertiary sector is
constantly rising. These factors increased the attention to this new working environ-
ment, previously considered safe.
There is a scientific evidence that the occupational exposure to cleaning agents
and disinfectant occurring in non-industrial indoor settings are associated with
asthma onset or exacerbation. Moreover, exposure to moulds in damp building,
especially in North America and Northern Europe, plays an important role in deter-
mining or exacerbating asthma. The role of other indoor occupational exposures,
especially ETS, is not completely clear but could contribute to the burden of work-
related asthma. In conclusion, because of the high socio-economical burden of
work-related asthma and the increasing number of subjects working in non-industrial
indoor environment, there is an urgent need of public health intervention to avoid or
reduce the exposure to risk factors associated with indoor air-related asthma. The
intervention must start during the design and planning phase of new buildings, to
prevent the release of toxic substance from building materials and furniture, as well
as to reduce the risk of mould contamination. Afterwards, attention must be paid to
the use of non-toxic cleaning agents and to keep the ventilation system working
properly. Workers health surveillance could play an important role in primary and,
overall, in secondary prevention. Information of workers, especially those involved
in professional cleaning, is very important to discourage dangerous habits, such as
mixing reactive and irritant chemicals, and to implement the use of personal protec-
tive equipment, when appropriate.
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Chapter 9
Combined Effect on Immune and Nervous
System of Aluminum Nanoparticles
Qiao Niu and Qinli Zhang
Abstract Aluminum is believed to be a neurotoxicant for a lot of years and thought

to be related with Alzheimers disease. In recent decades, aluminum nanoparticles
have been utilized widely in many fields, and their potential adverse effect on health
drew great concern. Al2O3 nanoparticles (ANPs) can be inhaled more deeply into
the respiratory system, and translocated into the bloodstream to induce immunotox-
icity and into the central nervous system to induce neurotoxicity, of which the pos-
sible mechanisms are summarized in this chapter. ANPs may induce pneumocyte
apoptosis by triggering oxidative stress and inhibit or activate activity of cytokines,
and the immunotoxicity induced by nanoalumina (Nano-Al) particles was higher
than that of macro-sized alumina particles. Besides though blood compartment by
which ANPs damage the blood-brain barrier, ANPs may enter into the central ner-
vous system through the olfactory nerve. ANPs impair behavioral performance of
model organisms and rodents. ANPs may induce neural cell death by triggering
apoptosis, necrosis, and autophagy, complicate cell signal transmission pathways,
and promote A deposit and degeneration.
Keywords Alumina nanoparticle Immunotoxicity Oxidative stress

Neurotoxicity Cell death Signal pathway
Q. Niu (*)
Occupational Health Department, Public Health School, Shanxi Medical University,
Shanxy, Taiyuan 030001, China
e-mail: niuqiao55@163.com
Q. Zhang
Shanxi medical University, Taiyuan 030001, China

DOI 10.1007/978-981-10-0351-6_9
116 Q. Niu and Q. Zhang
9.1 Wide Utilization of Aluminum Nanoparticles

and Concern on Their Toxicity
9.1.1 Toxicity of Aluminum
Aluminum is a very abundant metal element in the environment, comprising 8 % of

the earths crust and standing at third position in richness as elements. It is used
extensively in many fields of modern life, such as industry, medicine, food, trans-
portation, and living utensil, and may enter the human body from the environment
via air, diet, drinking water, food, cosmetic, or medication. Concerns about alumi-
num toxicity have persisted since the demonstration as a potential neurotoxicant by
Doellken more than 100 years ago [1]. This initial finding led to the extensive stud-
ies on increment of aluminum concentrations, senile plaque, and neurofibrillary
tangles in the brain tissues of patients with Alzheimers disease (AD) [2, 3]. Various
mechanisms have been proposed for aluminum-induced neurotoxicity, including
free-radical damage via enhanced lipid peroxidation and impaired glucose metabo-
lism, disturbed signal transduction and protein modification, alterations in the axo-
nal transport, and abnormal phosphorylation level of neurofilaments [46].
Aluminum is relatively stable in the form of alumina (aluminum oxide).
9.1.2 Chemical Property and Utilization of Nano-Al
Aluminum oxide, commonly called alumina, is a chemical compound of aluminum

and oxygen with the chemical formula Al2O3 and is the most commonly existing
type of several aluminum oxides, specifically identified as aluminum(III) oxide. It
occurs in its crystalline polymorphic phase -Al2O3, in which it constitutes the min-
eral corundum, a lot of kinds of which form the precious gemstones ruby and sap-
phire. Al2O3 is basically used to produce aluminum metal generally via electrolysis
process, as an abrasive owing to its hardness and as a refractory material owing to
its high melting point [7]. The nanosized form of aluminum oxide, Nano-Al, has
greatly enlarged its utilization in many more industries and fields, such as transpar-
ent ceramics, cosmetics, precision polishing materials, special glass, arms, aircraft,
electronics, dispersion strengthening, nanocomposites, constituent of rocket propel-
lant, constituent of explosive for military and for mini-bomb killing cancer tissue,
solar cell, and drug delivery [8]. As of October 2013, the number of consumer prod-
ucts of nanosized materials has increased to 1628 since 2005 [9], in which alumina
is among the most abundantly produced nanosized particles; the market volume is
predicted to 5000 tons only in China in 2016 [10]. With this in mind, there has been
a recent emergence of concern dealing with the potential for toxicity and the lack of
data to substantiate or dismiss these concerns [1113].
9 Combined Effect on Immune and Nervous System of Aluminum Nanoparticles 117
9.1.3 Elevation of Concern for Toxicity of Nano-Al
Aluminum oxide was deleted from the US Environmental Protection Agencys

chemicals lists in 1988, but its fibrous form is still on the EPAs Toxics Release
Inventory list, and toxicity of its nanosized particles is still far from being illumi-
nated. With so wide utilization of Nano-Al, many people, production workers,
and consumers are being exposed to alumina nanoparticles occupationally and envi-
ronmentally, and in living condition, concerns regarding their safety and potential
toxic effect have complicated their usage. Possible adverse effect of nanosized alu-
mina on human beings health has not been deeply investigated and elucidated, and
the study on aluminum nanoparticles-induced adverse effect is rarely seen.
Toxicological studies [12, 14, 15] have shown increased toxicity of nanoparticles
(<100 nm) compared to micrometer-sized particles of the same composition, which
has raised concern about the impact on human health from nanoparticles.
In vitro and in vivo studies have shown that ANPs have multisystem and multi-
organ toxicity on experimental animals, especially on the immune and nervous sys-
tem. Thereby, we summarize the immune and neurological adverse effect of
Nano-Al in this chapter based on the published data and our studies, even if the
investigations were sparse.
9.1.4 Nano-Al Is Inhaled into the Respiratory System

and Induces Oxidative Damage
Based on breathing air dynamics, nanosized particles can be inhaled more deeply
into the respiratory system than large particles and deposited on the surface of the
system. The lung tissue is considered the primary target organ for inhaled nanopar-
ticles. Xiaobo Li et al. [16] performed H&E and TUNEL staining to detect pathol-
ogy and programmed cell death in alumina nanoparticle (Al2O3 NPs)-exposed mice
lung tissue. They found an inflammation and red blood cells located in the pulmo-
nary mesenchyme; pneumorrhagia characterized by interstitial red blood cell distri-
bution; massive lymphocyte infiltration, especially the subpleural area; lung cell
degeneration; and massive bronchial epithelial cell apoptosis. By in vitro study
using human bronchial epithelial cell (HBE cell), they also found significant incre-
ment of apoptosis (2.24 0.17 %), increased activities of caspase-3 and caspase-9 in
Al2O3 NP-treated cells, indicating HBE cell apoptosis is initiated by the intrinsic
apoptotic pathway, marked damage to the mitochondrial membrane potential, sig-
nificantly increased cytoplasmic cytochrome c, increased reactive oxygen species
(ROS) level, and increased malondialdehyde (MDA). Moreover, they also found
that Al2O3 NPs significantly triggered downregulation of mitochondria-related
genes located in complex I, IV, and V. After having damaged epithelial cells of the
lung tissue, alumina NPs may be translocated from the respiratory system to other
organs and systems.
Direct input into the blood compartment from the lung tissue is certainly an
important translocation pathway of NPs in mammals. However, since predictive
particle deposition models indicate that respiratory tract deposits alone may be far
from fully accounting for the NP burden in the body, especially in the central ner-
vous system [17], we should consider as well input from other pathways.
9.2 Immune Toxicity of Nano-Al In Vivo and In Vitro Studies
9.2.1 Aluminum NPs and Alumina NPs Damage Alveolar

Macrophages and Pneumocytes with Immune Response
Alveolar macrophages are very important first frontier immune cells against foreign
materials which are inhaled into the respiratory system. In an in vitro study using
human alveolar macrophages (U937) and human type II pneumocytes (A549)
coculture treated with aluminum nanoparticle and Al2O3 NPs, Laura et al. [18]
found that the macrophages as frontier immune cells were more susceptible to the
NPs than the epithelial cells, but if the macrophages were not present, the pneumo-
cytes showed significant cell death, indicating that the macrophages actively engulf
exotic nanoparticles and interacted with toxicity of the NPs and protected the pneu-
mocytes. The main function of macrophages is to destroy foreign material via
phagocytosis. The authors assessed if the macrophages could still phagocytose
bacteria named community-acquired methicillin-resistant Staphylococcus aureus
(ca-MRSA) after treatment with the NPs and found that the Al2O3 NPs did not
impair phagocytosis of macrophages to the bacteria, but the Al-NPs did, meaning
that the Al-NPs alter the cell function and their higher immunotoxicity than the
Al2O3 NPs. While ca-MRSA was exposed to Al-NPs, no decrease in bacterial num-
bers was observed following overnight incubation, indicating that the Al-NPs do not
kill this specific strain of bacteria, and the reason of Al-NP-reduced phagocytosis
may be due to the Al ions released by aluminum nanoparticles, which chemically
alter the cellular environment and finally disrupt the phagocytic process. In a PCR
assay, Al2O3 NPs alone induced slightly the NF-kB pathway, but the Al-NPs did not
show this induction. ca-MRSA alone generated NF-kB pathway activation in cocul-
tured cells, but when the Al-NPs and Al2O3 NPs were present with ca-MRSA and
together treated the coculture, the cocultured cells did not generate activation of the
NF-kB pathway, indicating that the NPs are capable of altering or abolishing the
cells response to a pathogen via the NF-kB pathway. ca-MRSA infection alone
induced inflammatory markers interleukin (IL)-6 and tumor necrosis factor alpha
(TNF-) response in coculture; the NPs alone did not show this effect, but while the
cocultured cells were infected by ca-MRSA and the NPs were present, the expres-
sion of IL-6 and TNF- induced by ca-MRSA was abolished, showing that the NPs
inhibited ca-MRSA-induced IL-6 and TNF- expression. In the ELISA, Al-NPs
inhibited the secretion of IL-6, IL-8, IL-10, IL-1, and TNF- too, evidencing the
results of the PCR assay.
9.2.2 Alumina NP-Induced Immunotoxicity Is Complicated
In a repeated dose exposure experiment reported by Eun Jung Park et al. [19],
6-week-old male ICR mice were acclimatized for 1 week, and then Al2O3-NPs were
administered orally at a dose of 1.5, 3, and 6 mg/kg for 13 weeks, and the control
group was treated with autoclaved water. Blood (approximately 1.2 mL/mouse) was
collected from the saphenous vein for biochemical and hemogram analysis, and
then the mice were sacrificed, and the brain, thymus, lung, heart, liver, kidneys,
spleen, and testis were collected for histological examinations. The levels of aspar-
tate aminotransaminase (AST), alanine aminotransferase (ALT), and lactate dehy-
drogenase (LDH) in blood were significantly different between the Al2O3-NP-treated
group and the controls. Compared with the controls, the levels of AST, ALT, and
LDH decreased in the mice treated with 1.5 and 3 mg/kg Al2O3-NPs, but interest-
ingly, these levels were markedly elevated in the 6 mg/kg Al2O3-NP-treated mice. In
addition, with the same tendency, the accounted number of white blood cells
(WBCs) and the proportion of lymphocytes in the WBCs in the mice treated with
1.5 and 3 mg/kg Al2O3-NPs were decreased compared with the control group, while
those in the 6 mg/kg Al2O3-NP-treated group were significantly increased; the pro-
portion of eosinophils in the WBCs in the mice treated with 1.5 and 3 mg/kg Al2O3-
NPs was increased than in the control group, whereas that markedly decreased in
the 6 mg/kg Al2O3-NP-treated group. The levels of IL-1 and TNF- in the Al2O3-
NP-treated groups did not show significant change compared with the control group,
and granulocyte-macrophage colony-stimulating factor and transforming growth
factor were not detected at a significant level in all samples tested. However, the
levels of IL-6 and monocyte chemotactic protein-1 increased in a dose-dependent
manner. The results of Eun Jung Park et al. indicated that the immunotoxic effect of
Nano-Al is complicated.
9.2.3 Al2O3 Nanoparticle Has Higher Immunotoxicity

Than Micro-sized Alumina
In a 30-day Al2O3-NPs exposure study performed by Li Huan and colleagues [20],

70 healthy SPF ICR mice (3 months old) were treated with Al2O3 nanoparticle
(13 nm diameter) at 25 mg/kg bw, 50 mg/kg bw, and 75 mg/kg bw as an exposure
dose grade and Al2O3 nanoparticle (50 nm diameter) at 50 mg/kg bw and bulk Al2O3
at 50 mg/kg bw as comparison between nanosized alumina particle and micro-sized
alumina particle, by nasal instillation, three times daily, continuously for 30 days.
Superoxide dismutase (SOD) activity and glutathione (GSH) content in the spleen
tissue and thymus tissue of Al2O3 particle-treated mice decreased significantly com-
pared with blank and solvent controls, and in a dose-dependent and particle size-
dependent manner, i.e., the higher the dose was, and the smaller the particle size
was, the higher the SOD activity and GSH contents were. While MDA content in
the spleen tissue and thymus tissue of Al2O3 particle-treated mice increased signifi-
cantly compared with blank and solvent controls, and in a dose-dependent and par-
ticle size-dependent manner, the oxidative stress level was increased with increment
of doses administered with alumina nanoparticles and with decrement of particle
sizes. Inflammatory cytokines IL-1, IL-1, interferon- (IFN-), TNF-, IL-2, and
IL-10 contents in the spleen tissue and thymus tissue increased significantly, indi-
cating immune response in Nano-Al-treated mice was upregulated. The results of
this study showed that respiratory exposure to Al2O3 nanoparticle could initiate oxi-
dative stress and immune response more strongly than the controls and bigger Al2O3
particles, implying Al2O3 nanoparticle has higher immunotoxicity than micro-sized
alumina.
9.3 Neurotoxicity of Nano-Al
9.3.1 Nanoalumina Induces Neurobehavioral Impairment
In an in vivo study with male ICR mice [21], Zhang et al. compared the neurotoxic-
ity of Nano-Al and nano-carbon (Nano-C) as reference of the same particle size and
different chemical property and micro-alumina (Micro-Al) as reference of the same
chemical property and different particle size. The animals were inoculated intrana-
sally (i.n.) per day with Nano-Al, Nano-C, and Micro-Al at the dose of 100 mg/kg
bw as experimental groups, whereas another group of animals that received 0.9 %
saline were used as controls. The mice were sacrificed 10 days post-inoculation.
Tested with Morris water maze, treatment with Nano-Al dramatically lengthened
the escape latency of animals (Nano-Al vs. control and Nano-C p < 0.01, respec-
tively; Nano-Al vs. Micro-Al, p < 0.05). During the probe trial when the platform
was removed, the Nano-Al-treated mice spent significantly less time in the target
quadrant (Nano-Al vs. control, p < 0.01; Micro-Al vs. Control, p < 0.05) and exhib-
ited fewer platform crossings (Nano-Al vs. control, p < 0.01; Micro-Al vs. Control,
p < 0.05). In Nano-C-treated groups, both measurements were decreased but showed
no significant difference from those of the control group (Nano-C vs. control,
p > 0.05 for both parameters). In contrast, comparisons between Nano-Al- and
Micro-Al-treated groups indicated that mice treated with Nano-Al required longer
escape latency, spent less time in the target quadrant, and crossed the platform fewer
times (Nano-Al vs. Micro-Al, p < 0.05). In an in vivo study with mice exposed to
Nano-Al particles by the respiratory tract [22], Xin Zhang and colleagues reported
that only in female mice the neurobehavioral changes and especially depression-
like behavior appeared.
Yinxia Li et al. [23] observed effects of acute exposure to Al2O3 NPs and bulk
Al2O3 (micro-sized) on locomotion behaviors of nematodes. After exposure for 6 h,
the significant decreases in head thrashes (p < 0.01) and body bends (p < 0.01) were
observed in both nematodes exposed to 51203.9 mg/L of Al2O3-NPs and nema-
todes exposed to the same doses of bulk Al2O3. Nevertheless, both head thrashes and
body bends in Al2O3-NP-exposed nematodes were lower than those in bulk Al2O3-
exposed nematodes. Moreover, after exposure for 48 h, the similar but more deterio-
rated locomotion behaviors were observed. The authors further examined the 10-day
chronic neurotoxicity from 8.1 to 23.1 mg/L of Al2O3-NPs and same doses of bulk
Al2O3 exposure on locomotion behavior of nematodes and got the similar results as
the acute exposure experiment got. The authors concluded that Al2O3-NPs are more
neurotoxic than bulk Al2O3, implying that alumina nanoparticles possess higher
neurotoxicity than micro-sized alumina particles.
9.3.2 Inhaled Particles May Enter into the Central Nervous

System via Mainly Two Routs: Blood Circulation
and Olfactory Nerve
Chen Lei et al. [24] injected Alizarin Red S-labeled nanoalumina at the dose of 1.25
mg/kg into the mouse cerebral circulation via the carotid artery and detected the
brain endothelium and astrocytes by staining for factor VIII as endothelium marker
and GFAP as astrocyte marker, respectively. They found that Alizarin Red S-labeled-
nanoalumina particles were colocalized with the brain endothelium 1 h after injec-
tion and were also appeared in astrocytes surrounding the cerebral vessels.
Twenty-four hours after injection, Alizarin Red S-labeled-nanoalumina particles
were diffused in brain parenchyma close to astrocytes. The distribution pattern of
Alizarin Red S-labeled-nanoalumina particles in one week after injection was simi-
lar to that in 24 h after injection, indicating that nanoalumina particles passed the
blood-brain barrier, entered into the brain tissue, and accumulated in the brain and
not eliminated from the brain components. In order to examine how the nanoalu-
mina particles impaired the BBB, the author further assessed the effects of nanoalu-
mina on the levels of occludin and claudin-5, two types of important tight-junction
proteins that regulate integrity and barrier function of the brain endothelium, in
human cerebral microvascular endothelial cells (HCMECs). Following a 6-h expo-
sure to nanoalumina, a dose-dependent decrease in expression of occludin and clau-
din-5 expression was detected in HCMECs. Nanoalumina also decreased
tight-junction protein expression in vivo. A single dose of nanoalumina (1.25 mg/
kg) was administered to mouse via the carotid artery, a gradual decrease in occludin
for up to 30 days after injection was observed. Injection with 1.25 mg/kg nanoalu-
mina significantly elevated blood-brain barrier permeability at day 3 after injection,
and this effect was preserved for up to 30 days, indicating that only a single intra-
vascular nanoalumina exposure can damage the BBB and the effect can last up to 30
days, and even the exposure dose is not high.
Though there was not a report on other pathways of Nano-Al entering the brain
tissue except for blood circulation, there was a report on Nano-C entering the brain
tissue via the olfactory nerve. Oberdrster et al. [25] exposed rats with Nano-C
particles (36 nm) for 6 h, sacrificed the rats, and removed and examined the lungs,
cerebrum, cerebellum, and olfactory bulbs of exposed rats at 1, 3, 5, and 7 days after
exposure. The concentration of Nano-C particles in the lung tissue decreased from
1.39 g/g on day 1 to 0.59 g/g by day 7 after exposure, but the Nano-C particles in
the olfactory bulb significantly and persistently increased, from 0.35 g/g on day 1
to 0.43 g/g by day 7, implying that the olfactory nerve is a channel for Nano-C
entering into the brain tissue. Based on this fact, the authors concluded that the CNS
can be targeted by airborne nanoparticles and that the most likely mechanism is
from deposits on the olfactory mucosa of the nasopharyngeal region of the respira-
tory tract to subsequent translocation via the olfactory nerve. Depending on particle
size, more than 50 % of inhaled nanoparticles can be deposited in the nasopharyn-
geal region during nasal breathing. The authors estimated that approximately 20 %
of the nanoparticles deposited on the olfactory mucosa of the rat can be translocated
to the olfactory bulb and then into other parts of the brain tissue. The increases of
carbon nanoparticles in olfactory bulbs are consistent with studies in rodents that
demonstrated that intranasally instilled solid ultrafine elemental particle translo-
cates along axons of the olfactory nerve into the CNS [26].
9.3.3 Nanoalumina Induces Cerebrovascular Damage

via Autophagy
In an in vitro study performed by Lei Chen [24], HCMECs were treated with
Alizarin Red S-labeled nanoalumina (1 g/mL) for 6 h and stained with MitoTracker
Red, MitoTracker Green, and MDC (marker for autophagic vacuoles). Alizarin Red
S-labeled nanoalumina (1 g/mL, stained red) was detected in HCMECs following
a 2-h treatment. After a 12-h exposure, Alizarin Red S-labeled nanoalumina was
visible as aggregates close to clustered mitochondria stained by MitoTracker Green,
which indicates the loss of mitochondria membrane potential, and a notable increase
of MDC intensity indicates enhanced autophagy.
In a same in vivo study performed by Chen Lei [24] described in Sect. 4.2, nano-
alumina (1.25 mg/kg) was administered to mouse via the carotid artery, and 24 h
later, mouse brains were taken out and subjected to mouse autophagy PCR array,
and 84 autophagy-related genes were examined; 13 autophagy-related genes
increased more than twofold. Then two frequently used autophagy marker proteins
LC3 and p62 were also examined. Similar to the results obtained in HCMECs, in
which a notable increase of MDC intensity showed an increased autophagy, an
increase in LC3 and p62 proteins, which indicate autophagy too, was observed 24 h
after nanoalumina injection and remained elevated for as long as 30 days after nano-
alumina administration. Combining Chen Leis results, we could hypothesize that
Nano-Al particles preserve continuously in blood circulation compartment and

damage the BBB and are difficult to be discharged from circulation compartment
and the brain tissue.
The effects of nanoalumina on protein degradation were assessed in HCMECs
using DQ-BSA, a probe based on bovine serum albumin (BSA) labeled with red
fluorescent BODIPY TR-X dye (Invitrogen). Treatment with nanoalumina dose-
dependently elevated activity of DQ-fluorescence, and protein degradation activity
was correlated with the formation of acidic vesicles, consistent with autophagolyso-
somes stained with LysoTracker Green.
9.3.4 Nano-Al Induces Neural Cell Death
In the same in vivo study as in Sect. 4.1 with male ICR mice [21], Zhang et al. com-
pared the neurotoxicity of Nano-Al and Nano-C as reference of the same particle
size and different chemical property and Micro-Al as reference of the same chemi-
cal property and different particle size. The animals were inoculated intranasally
(i.n.) per day with Nano-Al, Nano-C, and Micro-Al at the dose of 100 mg/kg bw as
experimental groups, whereas another group of animals that received 0.9 % saline
were used as controls. The mice were sacrificed 10 days post-inoculation.
To investigate the potential mechanisms by which Nano-Al more strongly
impaired the neurobehavior of animals than Nano-C and Micro-Al did, the changes
in matrix metalloprotein (MMP) and ROS were observed. Treatment with 100 mg/
kg Nano-Al but not with the same concentration of Nano-C resulted in a highly
significant decrease in MMP. Alterations in mitochondrial potential may result in
induction of cellular oxidative stress; indeed, ROS production measurements
showed that Nano-Al led to a marked induction of oxidative stress.
Nano-Al-mediated MMP loss and significantly higher ROS production con-
firmed that Nano-Al may cause more severe neurotoxicity than Micro-Al. On the
other hand, Nano-C with the same nanoparticle size resulted in only mild neurotox-
icity that was not significantly different compared with controls treated with 0.9 %
saline.
Both Nano-C and Nano-Al at 100 mg/kg bw were toxic and enhanced the
necrotic rate, and the necrotic rate induced by Nano-Al was markedly higher than
apoptotic rate it induced. Apoptosis was also observed in Nano-Al-treated mice and
became more pronounced in Micro-Al-treated mice.
LC3 is a mammalian homolog of yeast Atg8, the only reliable marker of autopha-
gosomes that indicate autophagy process. In order to recognize if autophagy exists
in nanoparticle-induced neurotoxicity, the authors observed LC3 expression in the
study and found its expression was low.
Lower expression of LC3 in the study suggests that autophagy may not be the
major cell death mode in neural cells induced by Nano-C, Nano-Al, and Micro-Al.
Furthermore, robust caspase-3 activation likely indicates significant apoptosis in
Micro-Al-treated mice. The results also indicated that Nano-Al treatment led to
neural cell death; and necrosis may be a major cell death mode in nanoparticle-
induced neurotoxicity.
Furthermore, in an in vitro study performed by Zhang et al. [27], observed under
light microscope, neural cells treated with Micro-Al presented shrink and irregular
shape, indicating apoptotic-like cell death, while Nano-C particle-treated cells
exhibited blackish color, flat, and swelling shape in cell body, indicating necrotic-
like cell death. The Nano-C particles with the same size as Nano-Al could enter into
the cell body and accumulate in the cell cytoplasma. Whereas treated by Nano-Al
particles with the same chemical composition of Micro-Al and the same size with
Nano-C, the neural cells displayed morphological characteristics of both Nano-C-
and Micro-Al-treated cells, presenting both brownish particle accumulation and
condensed nuclei and cell organelle disrupture and necrotic cell death.
Under transmission electron microscope, margination of condensed chromatin
appeared in the Micro-Al-treated cells; the Nano-C-treated cells displayed nanopar-
ticles inside the cytoplasma and nucleus, indicating the disrupture of cellular and
nucleus membrane, while Nano-C-treated cells manifested the nanoparticles inside
the cytoplasma with disrupted cell membrane, chromatin aggregation, and broken
fragments surrounded by dissolved cytoplasma and organelles, indicating presenta-
tion of both the Nano-C- and Micro-Al-induced cell impairment features.
Endocytosis appeared in Nano-Al-treated cells, and the cell bodies and their nuclei
seemed to be disrupted and dissolved in the presence of the Nano-Al particles.
Nanoparticles of alumina are located in the primary lysosome, secondary lysosome,
and accumulated lysosomes in neural cells. The cell viability in Nano-Al-exposed
cells was much lower than those in Micro-Al- and Nano-C-exposed cells (p < 0.05,
p < 0.01). However, there was no significant difference between the viabilities of
cells treated with Nano-C and Micro-Al (p > 0.05).
9.3.5 Aluminum Nanoparticles Induce Activation of MAPK

Signal Pathway or Inhibit It
In a study performed by Jung-Taek Kwon [28], rats were exposed to Al-NPs by

nasal instillation at 1 mg/kg body weight (low exposure group), 20 mg/kg body
weight (moderate exposure group), and 40 mg/kg body weight (high exposure
group), for a total of three times, with a 24-h interval after each exposure. Inductively
coupled plasma mass spectrometry (ICP-MS) analysis indicated that the presence of
aluminum in the olfactory bulb and the brain of Al-NP-treated mice was increased
dose dependently. In microarray analysis, the regulation of mitogen-activated pro-
tein kinase (MAPK) activity was significantly overexpressed in the Al-NP-treated
mice than in the controls (p = 0.0027). Moreover, Al-NPs induced the activation of
ERK1 and p38 MAPK protein expression in the brain, but did not alter the protein
expression of JNK, when compared with the controls. The results demonstrate that
the nasal exposure of Al-NPs can permeate the brain via the olfactory bulb and
modulate the gene and protein expression of MAPK and its activity. But another
study confirmed that Al-NP exposure activated the JNK pathway [29].
But, in another study [30], the effect of Nano-Al on brain energy metabolism was
evaluated in alumina NP-treated and NP-untreated mouse brain homogenates via
western blot. The results indicated that Nano-Al inactivated MAPK and dephos-
phorylated it at Thr172 and reduced the expression of AMP-activated protein kinase
(AMPK) in the brain compared to the untreated mice, while the total AMPK level
remained unchanged. Similarly, the AMPK activity was also reduced in the brain
homogenates of Nano-Al-treated mice analyzed through the CycLex AMPK activ-
ity assay method. Additionally, the expression of p-AMPK was measured via immu-
nofluorescence in the hippocampal cornu ammonis 1 (CA1) and cortical regions of
nanoalumina-treated and nanoalumina-untreated mice. The images of immunofluo-
rescence revealed that nanoalumina significantly inhibited the expression of
p-AMPK, which supported western blot results, suggesting that alumina nanopar-
ticles are involved in the disturbance of brain energy metabolism.
9.3.6 Nano-Al Can Induce Oxidative Stress in Neural Cells

Both In Vitro and In Vivo
To analyze whether mouse hippocampal neural cell line, HT22 cells, can take up
Nano-Al and increase their aluminum level, morin staining was performed in the
same study as Sect. 4.5 [30]. Exposure of HT22 cells to Nano-Al caused an increased
uptake of Nano-Al, which ultimately increased the aluminum abundance in cultured
HT22 cells. Furthermore, to assess whether Nano-Al induces oxidative stress in
HT22 cells, 8-oxo-guanine (8-OxoG) staining was performed using an anti-8-OxoG
monoclonal antibody. The immunofluorescence images show that Nano-Al induced
oxidative stress and produced a significantly high number of ROS in the Al-NP-
treated HT22 cells in contrast to untreated HT22 cells.
In an in vivo study performed by the same authors, Nano-Al was peripherally
administered to ICR female mice for three weeks. The immunohistological evalua-
tions for abundance of brain aluminum were conducted in the hippocampal CA1,
CA3, and dentate gyrus (DG) and cortical regions of the female mouse brain. The
results indicated that exogenously administered nanoalumina significantly increased
brain aluminum abundance compared with the untreated control mice. In vivo
8-OxoG staining was performed to analyze the extent of oxidative stress induced by
nanoalumina, and it was evident from the immunostaining images that Nano-Al
induced oxidative stress by increasing 8-OxoG expression in the brains of Al-NP-
exposed mice. This trend was mainly observed in different parts of the hippocampus
including CA1 and CA3, respectively, and DG and the cortical regions in Al-NP-
treated mice, while no or only a few 8-OxoG appearances were seen in the hippo-
campus and cortical regions in the untreated control mice.
9.3.7 Nano-Al Can Induce A Production via Amyloidogenic

Pathway in Mice
The same authors of Sect. 4.6 study further to investigate A production via the
amyloidogenic pathway as a consequence of Nano-Al treatment in mice. The west-
ern blot results showed that the administration of nanoparticles to mice enhanced
the amyloidogenic pathway of A production. Nano-Al upregulated the expression
of the amyloid precursor protein (APP) and -secretase beta-site amyloid precursor
protein cleaving enzyme 1 (BACE1) activity, which significantly increased the gen-
eration of A in treated mice compared with untreated controls. Moreover, it also
caused downregulation of the -secretase enzyme sAPP-, which is responsible for
the generation of nontoxic A peptides through a non-amyloidogenic pathway. The
levels of soluble A142 in the brain homogenates were measured through the
ELISA method and revealed that Nano-Al significantly increased the production of
A142. Interestingly, Nano-Al also caused the formation of A aggregation and
plaques in the hippocampus and cortical regions of the brain in Al-NP-treated mice,
which was measured immunohistopathologically both via the A (6E10) antibody
and thioflavin S staining. The effect of increase in the A level produced by Nano-Al
on the hyperphosphorylation of microtubule-associated tau at ser413 and synapse
related proteins, including synaptophysin and postsynapse density protein 95 (PSD
95), was investigated via western blot. The result indicated that A induced a sig-
nificant increase in the expression level of p-tau (while the total tau protein level
was unchanged) and downregulated the expression of synaptophysin and PSD 95
proteins in treated mice compared to the controls.
9.3.8 Nano-Al Induced Neurodegeneration In Vivo
The toxic effect of the administered Nano-Al on inducing neurodegeneration was

further examined by Shahid Ali Shah, who analyzed the expression of apoptotic
markers via western blot. The results indicated that Nano-Al significantly upregu-
lated the expression of various apoptotic markers or apoptotic signals, such as
cleaved caspase-3 and cleaved PARP-1, in the hippocampus and cortical sections of
the mouse brain. However, caspase-3 and PARP-1 were less expressed in untreated
mice. Additionally, Fluoro-Jade B (FJB) staining was performed to investigate the
neurodegeneration in the hippocampus and cortical regions in mice by the adminis-
tered Nano-Al. The staining of the cortical and hippocampal CA1, CA3, and DG
regions of nanoalumina-treated mice revealed that the alumina nanoparticles
induced neuronal cell death, as was evidenced by the number of positive FJB cells,
whereas no such positive stainings were observed in the brain sections of the control
mouse.
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29. Kleinmana MT, Araujob J, Nelc A, Sioutasd C, Campbelle A, Conga PQ, Lia H, Bondya
SC. Inhaled ultrafine particulate matter affects CNS inflammatory processes and may act via
MAP kinase signaling pathways. Toxicol Lett. 2008;178(2):12730.
30. Shah SA, Yoon GH, Ahmad A, Ullah F, Ul Amin F, Kim MO. Nanoscale-alumina induces
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2015;7:15225.
Chapter 10
Non Pulmonary Effects of Isocyanates
Paola Pedata, Anna Rita Corvino, Monica Lamberti, Claudia Petrarca,

Luca Di Giampaolo, Nicola Sannolo, and Mario Di Gioacchino
Abstract Isocyanates are highly reactive compounds of low molecular weight con-
taining the functional group N=C=O. Isocyanates are increasingly used in the fab-
rication of many products such as elastomers, paints, adhesive, coating, insecticides,
and resins with a variety of industrial applications after polymerization with alco-
hols to form polyurethanes. The high chemical reactivity of isocyanates, an impor-
tant characteristic in industrial applications, also makes them toxic products.
Isocyanates currently are the most commonly identified cause of occupational
asthma in industrialized countries. Exposure to diisocyanates, polyisocyanates, and
polyurethane additives may also cause allergic and irritant contact dermatitis (ACD,
ICD) and may result in neurotoxic and carcinogenic effects.
The purpose of this paper is to review and synthesize the literature regarding
non-pulmonary health effects of isocyanates to address several key unresolved
P. Pedata (*) A.R. Corvino M. Lamberti N. Sannolo

Department of Experimental Medicine, Section of Hygiene, Occupational Medicine and
Forensic Medicine, Second University of Naples, Naples, Italy
e-mail: paola.pedata@unina2.it
C. Petrarca
L. Di Giampaolo
Chieti, Italy
M. Di Gioacchino

DOI 10.1007/978-981-10-0351-6_10
130 P. Pedata et al.
issues, including the carcinogenic and neurotoxic effects, and to focus on the often
unrecognized skin effects.
Keywords Isocyanates Health effects Carcinogenic effects Neurotoxic effects

Skin effects
10.1 Introduction
Isocyanates are highly reactive compounds of low molecular weight containing the
functional group N=C=O.
Isocyanates are classified, based on number of N=C=O groups in the molecules,
into monoisocyanates (one NCO), diisocyanates (two NCO), or polyisocyanates
(multiple NCOs). In diisocyanate, the two functional groups can directly polymer-
ize with alcohols to form polyurethanes, resins with a variety of industrial applica-
tions. Polyisocyanates represent the major source of exposure to isocyanate groups
in many workplaces, and like the diisocyanate monomers from which they are
derived, such as toluene diisocyanate (TDI), diphenylmethane diisocyanate (MDI),
and hexamethylene diisocyanate (HDI), they are increasingly used in the production
of elastomers, paints, adhesives, coatings, insecticides, and many other products.
Aliphatic isocyanates such as those based on HDI are used mostly in external
paints and coatings because of their excellent resistance to chemicals and abrasion
and superior weathering characteristics such as gloss and color retention.
Aromatic isocyanates, such as MDI, are used in many applications such as
foams, adhesives, sealants, elastomers and binders, which require fast curing rates
and have less stringent requirements on their chemical and mechanical stability.
Polyurethane foams are a major end use of aromatic isocyanates [1]. There are
several reports of sensitization to polyurethane products also in a domestic setting,
such as to a plastic watchstrap, spectacle frames, and a pacemaker lining due to the
increasing popularity of do it yourself; for this reason, isocyanates are likely to
become more common chemicals within houses [2].
The high chemical reactivity of isocyanates, an important characteristic in their
industrial use, also makes them toxic. Despite substantial research on isocyanates,
the pathogenic mechanisms, host susceptibility factors, and dose-response relation-
ships remain unclear [35]. Isocyanates can bind to carrier proteins, via the reaction
of the NCO group with nucleophiles such as SH, NH2, NH, and OH groups present
on these proteins. Several peptides and proteins found in airway epithelial cells,
serum, and skin have been observed to bind diisocyanates, including glutathione
[6, 7], albumin [8, 9], tubulin [10], and keratin [9, 11]. Covalent binding of isocya-
nate groups to carrier proteins is likely an important step in the chain of events lead-
ing to health effects, in particular sensitization and asthma [1].
Respiratory exposure to isocyanates has long been considered the primary way of
exposure, and thus, research, regulation, and prevention have focused almost exclu-
sively on airborne isocyanate exposures [12]. However, respiratory exposures have
10 Non Pulmonary Effects of Isocyanates 131
been reduced through improved hygiene controls and the use of less-volatile isocya-
nates [1], thus potentially increasing the relative importance of skin exposure. Numerous
isocyanate end uses, such as spraying and application of foams and adhesives, may
cause isocyanate skin exposure from deposition of aerosols and/or absorption of vapors.
Typical workplace isocyanate exposure levels are not irritating and give few warning
signs and skin protective equipment may not be worn, even when respiratory protection
is used [13]. Skin exposure is the consequence of direct contact of unprotected skin or
the failure of personal protective equipments, like gloves; particularly events such as
spills, cleanup, and contact with contaminated equipments represent the major oppor-
tunities for isocyanate skin exposure. There are some investigations showing that iso-
cyanates [14] and solvents [15] can be detected underneath gloves.
Isocyanate skin exposure could contribute a significant part of the total body
burden. Multiple lines of evidence from animal studies and clinical, epidemiologic,
and biomarker studies, as well as anecdotal evidence, suggest that in certain expo-
sure setting, human skin likely is an important route of isocyanate exposure and can
contribute to the development of isocyanate health effects [16].
Isocyanates currently are the most commonly identified cause of occupational
asthma in industrialized countries, where its prevalence among exposed workers
ranges from 1 % to even 25 %. These chemicals were also reported as causal factors
of other respiratory disorders as nonobstructive bronchitis, rhinitis, chronic obstruc-
tive pulmonary disease, and less commonly extrinsic allergic alveolitis [16, 17].
Several observations suggest that skin exposure occurs and can contribute to the
development of isocyanate asthma presumably by inducing systemic sensitization.
In fact, isocyanate respiratory exposure alone, without any skin exposure, seems
unlikely in most work settings. Isocyanate asthma occurs in settings with minimal
documented respiratory exposures but clear potential for skin exposure, and splashes
and spills have been reported by workers who subsequently develop isocyanate
asthma [1822].
Exposure to diisocyanates, polyisocyanates, and polyurethane additives may
also cause allergic and irritant contact dermatitis [23]. Contact hypersensitivity
(allergic contact dermatitis) following skin exposure to isocyanates is well docu-
mented in animals and in clinical dermatologic literature, with sensitization con-
firmed with patch testing [24, 25]. Allergic contact dermatitis has been reported
following skin exposure to isocyanates and polyurethane products in a number of
different workplace and non-occupational settings, but has not been considered
common and is rarely reported in workers with isocyanate asthma [24, 2628].
However, allergic contact dermatitis may be more common than suspected because
symptoms can be mild, workers being evaluated for asthma are frequently not asked
about skin problems, and patch testing can be falsely negative [24, 29].
Another potential health disease related to exposure to isocyanates are neuro-
toxic effects consisting in lightheadedness, headache, insomnia, mental aberrations,
impaired gait, loss of consciousness, and coma due to acute exposure and alterations
in the central and peripheral nervous systems for chronic exposure. Moreover, a
systematic review of the literature evaluating the causal association on humans does
not exist to support this alleged association [30].
Another open question is whether occupational isocyanate exposure is a carcino-

genic hazard. TDI has been classified as carcinogenic in animals on the basis of
gavage administration studies, but no conclusions are available on inhalation expo-
sure. TDI is classified as a Group 2B carcinogen (possibly carcinogenic to humans)
by the International Agency for Research on Cancer (IARC) [31] and as an A4
carcinogen (not classifiable as a human carcinogen) by the American Conference of
Governmental Industrial Hygienists (ACGIH) [32]. For MDI, there is suggestive
evidence for carcinogenicity in rats. Both chemicals have been positive in a number
of short-term tests inducing gene mutations and chromosomal damage [33].
Over the past several years, there has been an increasing collection of clinical
and epidemiologic data related to isocyanate health effects, especially those regard-
ing respiratory ones, although knowledge and awareness remain limited regarding
the non-pulmonary effects of isocyanates and some clinical studies are still unrec-
ognized. Our purpose in this paper is to review and synthesize the literature regard-
ing non-pulmonary effects of isocyanates to address several key unresolved issues,
including the carcinogenic and neurotoxic effects of isocyanates, and to focus on the
often unrecognized skin effects.
10.2 Literature Search
We selected the most relevant contributions to the literature in clinical and epide-
miologic fields starting with the information retrieved from PubMed (http://www.
ncbi.nlm.nih.gov/pubmed/), Google Scholar (http://scholar.google.com), and
ScienceDirect (www.sciencedirect.com) using the following keywords: isocya-
nates OR diisocyanates OR MDI OR TDI OR HDI, AND health effects,
AND skin disease OR sensitization, AND cancerogenesis AND neurotoxic-
ity, and other synonymous terms and our own extensive collection of isocyanate
publications. Additional papers were identified from the reference lists of the
selected relevant articles.
The search yielded about 110 articles which were further reviewed; at the end of
this selection process, 82 articles were deemed relevant to this review and were
examined with a particular emphasis on non-pulmonary effects of isocyanates, such
as skin diseases and neurotoxic and carcinogenic effects.
10.3 Health Effects
10.3.1 Skin Diseases
The isocyanates are an important cause of occupational asthma, but descriptions of

ACD and ICD due to them are relatively uncommon [24]. In a mouse local lymph
node assay examining the dermal sensitization potential of TDI, Woolhiser et al.
[34] reported irritation at the TDI application site [34]. Second, Arnold et al. [35]
reported that the animal studies are insufficient to characterize the dermal irritation
potential of TDI due to the lack of documentation and use of nonstandard methodol-
ogy; the overall weight of evidence indicates that TDI is irritating to the skin of
experimental animals [35].
ACD caused by isocyanates has been reported mainly in connection with occu-
pational exposure in the manufacture of polyurethane products used in the plastics,
car and textile industries, flooring, and the manufacture of medical, electronic, and
foam products. It has also been reported in sculptors [36] and a molders [37],
although there is still limited knowledge about the skin diseases in occupational
setting because the fact that most studies to date have been cross-sectional in design,
small in size, or based on selected clinical cases or production workers rather than
end use workers.
One of the first investigations on the skin effects of polyurethane products is that
of Emmet et al.; this study reported skin rashes on exposed skin areas in eight work-
ers of polyurethane molding plant, with positive patch test reactions to
dicyclohexylmethane-4,4-diisocyanate (DMDI, synonymous with hydrogenated
methylene-4,4-diphenyl diisocyanate HDI) and diaminodiphenylmethane (MDA)
in two of them [38]. White et al. showed ACD and ICD due to DMDI in uncured
resin in a factory of car badges [39]. Frick et al. demonstrated ACD in workers of a
company manufacturing flooring laminate boards, after the introduction of a water-
repellent lacquer based on MDI. Five workers, engaged as machine operators where
lacquer was sprayed onto the boards, developed eczematous lesions of the forearms,
hands, or arms. Patch testing showed positive reactions to MDA in four, to MDI in
one, to HDMI in one, and to a lacquer in three of them [40]. Another study of Frick
et al. reported severe eczema in 17 workers exposed to glue based on DMDI, at a
factory manufacturing medical equipment. Contact allergy to DMDI, other isocya-
nates, and/or MDA was demonstrated in 13 individuals [27].
Isolated cases of ACD in workers exposed to MDI were also reported in different
occupational environment. Hannu et al. demonstrated a case of ACD due to MDI
and MDA from accidental occupational exposure in a female worker of a manufac-
turing plant of electronic components [41]. Schroder reported a case of ACD to
MDI, present in polyurethane adhesive, TDI, MDA, and epoxy resin in a female
engaged in a plant manufacturing grinding tools [42]. Estlander et al. diagnosed
occupational contact dermatitis in three workers exposed to MDI present, respec-
tively, in a hardener of core binder, a hardener of adhesive, and a laboratory mixture
of diisocyanates [43]. Lidn reported a case of ACD of the forearms in a molder
exposed to MDI-containing product in a hospital [37]. Tait and Delaney reported a
case of a maintenance fitter who developed ACD after cleaning filters contaminated
with aptane isocyanate based on MDI [44]. Kerre reports a patient who developed
an acute allergic contact dermatitis using a DMDI-charged cartridge to create resin-
coated 3D labels within an office environment [45].
According to Aalto-Korte et al. who analyzed 54 cases of ACD due to mono-
meric isocyanates, motor vehicle industry was among the most significant occupa-
tional fields for isocyanate contact allergy. They identified many sources of allergy
to MDI such as polyurethane foam used in the production of car insulation material,
pistol foam containing MDI, and uncured polyurethane insulation material [46]. A
recent study of Kiec-Swierczynska et al. in a vehicle equipment factory revealed
work-related contact dermatitis in 12 workers exposed to polyurethane foam con-
taining MDI. Seven of them developed contact allergy to MDA and were diagnosed
with occupational ACD. Irritant skin reactions to the antiadhesive agent were also
observed, in three cases coexisting with occupational ACD [47].
There are several reports of sensitization to polyurethane products also in domes-
tic setting, such as to a plastic watchstraps [26], spectacle frames [48], a pacemaker
lining [49], and do-it-yourself products used for renovating objects [2], though only
one of these reports has been specifically identified, sensitization to 1,6-hexamethylene
diisocyanate. It is also described a case of allergic contact dermatitis caused by
isocyanates, specifically DMDI, in resin jewelry making. Furthermore, this is not
occupational related [50].
Isocyanates causing allergy contact dermatitis included MDI [24, 37, 4143],
TDI [24], HDI [24, 51], and DMDI [27, 39, 45, 52, 53]. Allergy contact dermatitis
due to MDI was seen more frequently than that related to other isocyanates, which
may be a consequence of the fact that MDI represents more than a half of the world-
wide isocyanate production [46].
Occupational allergic contact dermatitis usually develops after months or years
of exposure. However, strong allergens may sensitize after a single exposure [54].
According to Kanerva et al., one can assume that sensitization from a single expo-
sure has taken place when a patient, with no previous eczema, develops the first skin
symptoms soon after accidental exposure and patch testing with the chemical pro-
vokes an allergic reaction.
The most common locations of occupational dermatitis are generally the hands
and forearms, but the face is also commonly affected. In a report from a general
dermatology clinic, facial symptoms were more common among TDI-positive or
isophorone diisocyanate (IPDI)-positive cases than among MDA-positive or MDI-
positive ones [55]. Although in later investigations no difference was noted when
MDI-positive patients were compared with IPDI-positive or TDI-positive cases,
facial symptoms resulted not so common in patients reacting only to MDA [46].
Moreover, cases of urticaria due to MDI were demonstrated [5658]. Particularly,
Stingeni et al. reported a case of nonatopic man with breathing difficulties for
3 months and urticaria on his face (mala and mandibular areas) for 2 months who
worked in a chemical factory manufacturing adhesives and 1 year previously had
been assigned to the mixing of polyurethane glues. The symptoms developed a few
minutes after every working exposure to the Isonate M143 glue containing
diphenylmethane-4,4-diisocyanate (MDI). This study is the first report of concomi-
tant type I and type IV sensitivities to MDI, as respectively shown by the immediate
urticaria-type patch test reaction to Isonate M143, positive radioallergosorbent test
to MDI, and delayed positive patch test to MDI and Isonate M143 serial dilutions.
Contact urticaria belongs to the class of immediate skin immune response, as also
proved by specific IgE antibodies against MDI detected in the patient.
10.3.2 Neurotoxicity
Limited number of studies suggests potential neurotoxic effects from exposures to

isocyanates; acute exposure to high levels of TDI vapor has been associated with
lightheadedness, headache, insomnia, mental aberrations, impaired gait, loss of
consciousness, and coma, while chronic exposures have been associated with altera-
tions in the central and peripheral nervous systems.
One of the first case reports, conducted in 1987, described the accidental expo-
sure of three wharf workers at TDI during an accidental chemical spill. Compared
with 2 months postexposure, at 16 months postexposure, Full Scale Intelligence
Quotient (IQ) dropped an average of 23 points. Results from additional neuropsy-
chological testing at 16 months postexposure indicated severe deficits in all three
subjects in memory, manual dexterity, visuomotor tracking, mental flexibility, abil-
ity to detect figure-ground relationships, and word fluency [59].
Reyde et al. reported results of neuropsychological functioning of five men suf-
fering alleged physical, cognitive, and behavioral changes following exposure to
MDI. All workers reported experiencing subjective symptoms consisting of respira-
tory distress, headaches, depression, irritability, decreased calculating ability, and
reduced concentration. Wechsler Adult Intelligence Scale-Revised IQ revealed in
four of the five subjects weakness on the digit symbol subtest and in attention-
concentration testing. The authors concluded that neuropsychological test data sup-
port the presence of behavioral and cognitive correlates of CNS injury following
exposure to MDI [60].
Recently, Moshe et al. described a case report of a 60-year-old Israeli painter/
artist with central and peripheral neuropathic findings. His work was unusual in that
he painted large posters with different mixtures of organic solvents, including tolu-
ene, xylene, benzene, methyl ethyl ketone, TDI, acetone, and thinner. He did not use
any protective gloves and did not wear a mask. After 30 years as a painter, he devel-
oped weakness and paresthesia of the hands and feet, intention tremor, and diffi-
culty concentrating with memory deficits. He had mild atrophy and distal weakness
of both upper and lower limbs and demonstrated bradykinesia. He was evaluated
with several methods and was diagnosed as having peripheral and central neuropa-
thy, including ototoxic hearing loss because of long exposures to organic solvents
[61].
In recent years, a systematic review conducted by Hughes et al. demonstrated a
lack of quality epidemiological studies in the literature on neurotoxic health effects
and exposure to diisocyanates. The available evidence consists mostly of case
reports and case series, some of which are listed above. Using the Hill criteria or
considerations for causality, the authors found limited evidence for strength of asso-
ciation and consistency [30].
Thus, no robust and confirmed evidence of any primary peripheral or central
nervous system neurotoxic effect from diisocyanates has been reported in experi-
mental animal studies. Two-year inhalation studies on both MDI and TDI have been
conducted, in which daily observations of clinical signs were made. There were no
reports of clinical signs indicative of neurotoxicity [62, 63]. In short-term investiga-

tory studies conducted at higher exposure concentrations than the chronic studies,
clinical signs related to respiratory irritation were reported with no indications of
neurotoxic effects of MDI or TDI [6466]. Finally, one sub-chronic study with HDI
included a neurobehavioral testing segment, which was negative for such effects
[67]. Overall, the animal toxicology test data give no indication of neurotoxic effects
from diisocyanates.
10.3.3 Cancerogenesis
As well as to the neurotoxic effects, the carcinogenic risk of TDI and MDI exposure
remains an unanswered question.
TDI is classified as a Group 2B carcinogen (possibly carcinogenic to humans) by
the IARC [31], as a Category 2 carcinogen (suspect human carcinogen) in the
European Commission [68] [68], as reasonably anticipated to be a human carcino-
gen by the US National Toxicology Program (NTP) [69], and as an A4 carcinogen
(not classifiable as a human carcinogen) by ACGIH [32]. These classifications are
based on the increased tumor incidences observed by the NTP [70] when TDI in
corn oil was administered directly into the stomach of rodents by oral gavage [70].
Oral administration of commercial-grade TDI (analyzed as 85 % 2,4 isomer and
15 % 2,6 isomer) by stomach tube caused hepatocellular adenoma in female rats and
mice, fibroadenoma of the mammary gland and islet-cell adenoma of the pancreas
in female rats, and acinar-cell adenoma of the pancreas in male rats, while no
treatment-related tumors were observed after inhalation exposure of rats and mice.
However, this study was flawed both technically (i.e., mishandling of the test mate-
rial) and conceptually (i.e., gavage exposures) resulting in the formation of toluene
diamine, a known animal carcinogen, both prior to and after TDI administration
[35].
A long-term inhalation study with TDI was carried out by the International
Isocyanate Institute in Sprague-Dawley CD rats and CD-1 mice; in this study,
groups of male and female rats and mice were exposed to 0.05 and 0.15 ppm of TDI
by inhalation for 6 h/day, 5 days/week for 2 years. Authors concluded that type and
incidence of tumors and the number of tumor-bearing animals of either species did
not indicate any carcinogenic effect [63]. However, this study was considered inad-
equate for the evaluation of TDI carcinogenicity by the WHO. The major criticism
was that the TDI concentrations used were of magnitudes below acute LC50 values
for rats and mice [33, 71]
Regarding MDI, IARC has considered that there is limited evidence in experi-
mental animals for the carcinogenicity of a mixture of monomeric and polymeric
MDI. Industrial preparation of MDI was not classifiable as to its carcinogenicity to
humans (Group 3) [72].
Experimental studies on rats exposed to MDI by the inhalation route showed that
the exposure to polymeric MDI aerosol (a mixture containing 47 % monomeric
MDI and 53 % polymeric MDI) induces severe olfactory epithelium degeneration in

females and male Wistar rats at the highest concentration used; at the highest dos-
age, pulmonary adenomas were observed in males (6/60) and females (2/59) and a
pulmonary adenocarcinoma in males (1/60) [62]
In contrast to the study of Reuzel et al. in a long-term carcinogenicity study con-
ducted in female Wistar rats exposed by inhalation to MDI in aerosol form to 0.23,
0.70, and 2.06 mg/m3-), only 1 of 80 animals of the high-exposure group exhibited
a bronchoalveolar adenoma [73].
The carcinogenic risk of TDI and MDI exposure remains an unanswered ques-
tion. TDI was considered a possible human carcinogen, based on the sufficient evi-
dence of carcinogenicity in animals after oral administration; however, workers are
primarily exposed to TDI via inhalation, and it is important to note that toxicity
spectrum of TDI metabolites is likely to differ between the routes of
administration.
Many studies concluded that no risk could be associated with the inhalation of
TDI. Doe and Hoffman assessed the carcinogenic risk of TDI for humans by extrap-
olating data from the experimental studies in rats and mice and calculating cancer
potency. The study concluded that no risk could be associated with the inhalation of
TDI [74]. Some epidemiological studies with updates representing the combined
long-term mortality experience of more than 17,000 polyurethane foam production
workers failed to find an association between occupational exposure to diisocya-
nates and an increased risk of cancer [7580]. According to Bolognesi et al., the
available human evidence and the experimental data are inadequate to evaluate the
carcinogenic risk of isocyanates for humans [33].
Considering the wide range of uses of these compounds and the high number of
people occupationally exposed, further experimental and epidemiological investiga-
tions on the carcinogenicity of TDI and MDI are urgently needed.
10.4 Conclusion
Isocyanate exposures are common in todays workplace and can cause sensitization
and asthma representing an important health risk to workers. Moreover, there is still
limited and conflicting knowledge about neurotoxic and carcinogenic effects.
Respiratory exposure to isocyanates has long been considered the primary way of
exposure. However, respiratory exposures have been reduced through improved
hygiene controls and the use of less-volatile isocyanates, thus potentially increasing
the relative importance of skin exposure. Despite substantial research on isocya-
nates, the pathogenic mechanisms, host susceptibility factors, and dose-response
relationships remain unclear. Further clinical, epidemiological, and animal research
is needed to better understand isocyanate exposure risk factors and elucidate disease
mechanism, whereby, preventing workers exposure to isocyanates represents a
critical step in eliminating the health hazards associated with isocyanates. Applying
engineering controls, such as closed systems or mechanical ventilation, and
requiring personal protective equipment can help limit worker exposure to isocya-
nates. The use of chemical-resistant clothing and gloves is essential to protecting
workers skin from having contact with isocyanates, and specific types of personal
protective equipment (PPE) should be selected according to the hazard assessment
results of each workplace.
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Chapter 11
Skin Exposure to Nanoparticles and Possible
Sensitization Risk
Francesca Larese Filon
Abstract Due to the increased production and use of nanoparticles (NPs), there are
workers and consumers that can be exposed to NPs. There is a debate among scien-
tists to define possible effects related to this exposure and there are more open ques-
tions. The review evaluates the recent knowledge on this topic trying to classify NPs
in relation to their hazard for skin exposure, both for workers and consumers. While
the same kind of NPs can be safe for skin contact (such as titanium dioxide and zinc
oxide), others can exert a sensitization effect such as NPs that can release sensitiz-
ing metals (i.e., Ni, Pd, Co), as well as a toxic effects for NPs that can release toxic
metals such as Cd or As.
Due to the high surface/mass ratio, NPs can release more metals than bulk mate-
rials, increasing the risk of skin or systemic effects after the skin contact with them.
Moreover, NP size and the impairment of the skin barrier of exposed workers and
consumers are crucial points to be evaluated because they both can contribute to
NPs exposure and skin absorption.
Labeling is needed for NPs and products containing sensitizing or toxic metals
to advise users to protect them from direct contact with the skin.
Keywords Nanoparticles Skin exposure Skin absorption Allergic

sensitization
11.1 Introduction
Engineered NPs production is growing in quantity for industrial application [1]. The
National Science Foundation has estimated that by 2020, nanotechnology will
employ six million workers [2, 3]. Moreover, many workers and consumers are
handling nano-embedded products that are already available on the market. Many
F. Larese Filon (*)

Unit of Occupational Medicine, Department of Medical Sciences, University of Trieste,
Via della Piet 19, 34129 Trieste, Italy
e-mail: larese@units.it

DOI 10.1007/978-981-10-0351-6_11
144 F. Larese Filon
cosmetics contain NPs, mainly titanium dioxide and zinc oxide, due to the better
properties of creams. Many textiles, wound dressings, deodorants, antiperspirants,
pigments, varnishes, electronic devices, catalytic converters, and fuel cells are pre-
pared using NPs and can release NPs during their use. NPs can be released from
nano-enabled products, such as pastes, paints, glues, etc. and are potential sources
of dermal exposure to NPs [47].
NPs may exhibit new physicochemical properties due to the small size (1100
nm) with a faster and more efficient penetration into the human body than bulk
materials. Their high surface/mass ratio can cause a higher release of toxic sub-
stances compared to non-nanopowders.
Many studies have demonstrated the safe profile of NPs used in cosmetics [8, 9],
and the widespread use without any reported adverse effect can confirm that these
kinds of NPs did not harm the skin or the body. Different questions arise for other
NPs that are present in objects or other products that can have a possible local effect
or can penetrate the skin barrier with local or systemic effects [1012]. As different
NPs cannot be considered in the same way, we need to evaluate different classes of
NPs to define their hazard related to skin contact [12]. Data are scanty on human/
worker exposure, but recently a worker exposed to nano-nickel developed contact
dermatitis and occupational asthma [13] handling nanoscale nickel NPs without
protection measures that are already suggested for workers exposed to NPs [1, 2].
She worked with nanoscale nickel without a local exhaustion system nor in a glove
box, and she did not wear a respiratory mask that can protect exposed workers [14,
15]. Nanosized metallic nickel (Ni) has special properties, i.e., high surface energy,
high magnetism, and high surface area which makes it ideal for a number of indus-
trial processes [16]. There are some data to indicate that nanosized metallic nickel
(20 nm) is more toxic than standard-sized nickel (5 mm) in a rat model of lung
toxicity [17]. Crosera et al. [18] evaluate nickel NP skin absorption through human
skin using an ex vivo approach finding that these NPs applied on skin surface cause
an increase of nickel content into the skin and a significant permeation flux through
the skin, higher when a damaged skin protocol was used. They stated that preventive
measures are needed when NiNPs are produced and used due to their higher poten-
tial to enter in our body compared to bulk nickel.
Moreover, it is advisable to implement surveillance as NP and nano-embedded
products become more pervasive in the workplace [1921].
The aim of this review is to evaluate available literature on skin exposure to NP
to define aspects to be considered for risk assessment, with special attention for NPs
containing sensitizing metals.
11.2 Material and Methods
Literature were evaluated using the terms NP skin exposure, nanomaterial skin
exposure, irritant contact dermatitis epidemiology, and allergic contact derma-
titis epidemiology in PubMed, Thomson Reuters Web of Science, and Scopus
11 Skin Exposure to Nanoparticles and Possible Sensitization Risk 145
databases. Governmental institution guidelines (NIOSH) were used to insure a prac-

tical approach to workers protection. A total of 293 were selected and 54 were used
to elaborate this review.
11.3 Results
11.3.1 When Skin Exposure to NPs Is Possible?
People can be exposed intentionally, because they apply on the skin products that
contain NPs, such as creams, cosmetics, and wound dressing. Others can wear tex-
tile embedded with silver NPs or they touch objects coated with nano-products.
Workers can be exposed non-intentionally because they are producing, using, man-
ufacturing, handling, and processing NPs or products that can release NPs. Also
during the use of products containing NPs, it is possible a release from objects,
varnishes, glues, or other products that contain NPs. Since it is not compulsory the
labeling of products containing NPs, there are many commercially available prod-
ucts that are made using also NPs and that can come in contact with the skin. The
European Agency for Safety and Health at Work reported that the biggest risk of
exposure to NPs were in construction, health care, energy conversion and use, auto-
mobile (and aerospace) industry, chemical industry, and electronics and communi-
cation [22]. Table 11.1 reports a non-exhaustive list of industries/products.
11.3.2 Relevant Aspects for Skin Exposure to NPs
11.3.2.1 Source Domains
Dermal exposure can be evaluated using the conceptual framework elaborated by

Schneider et al. [23]. The source of contamination is NPs produced or released from
objects. From the source domains, the NPs can deposit on the skin or on other sur-
faces (direct contact). NPs can be transported to the air zone, and from there they
can contaminate the skin and surfaces by deposition. Protective gloves and aprons
can be contaminated themselves and resuspension of NPs can happen. Finally NPs
can be transferred to other surfaces and to the perioral region causing inadvertent
intake and ingestion. More information on risk resulting from skin contact can be
found in BauA [24].
Van Duuren-Stuurman et al. in 2008 [25] studied dermal exposure to NPs in ten
European enterprises using an observation model (Dermal Exposure Assessment,
DREAM). In the majority of tasks observed (39/45), dermal exposure cannot be
excluded, and the main routes for exposure were transfer and direct contact.
146 F. Larese Filon
Table 11.1 Examples of industries where skin exposure to NPs is possible (non-exhaustive)
Material Potential for skin
Industries handled exposure
Production of nanomaterials NPs Yes
Production of nanocomposites NPs Yes
Pharmaceutical industry NPs, liquid Yes
Toner production NPs Yes
Food production Liquid Yes
Tattoo ink production and application NPs Yes also inside the skin
Plant protection fertilizers Liquids Yes
Production and use of concrete Powders Yes
Paint production Liquid and Yes
powders
Ink production Liquid Yes
Cosmetics production Powders Yes
Coating application in textiles, plastics, cleaning, Liquid, powders Yes
health care, homecare
Electronic production Liquid, powders Yes
Battery production Liquid, powders Yes
Black rubber production Powders Yes
Sport industry Powders Yes
Fuel production Liquid Yes
Waste treatments All Yes
11.3.2.2 NP Characteristics
NPs emitted from processes or released from objects or textiles agglomerate and
settle on the skin and on surfaces. Therefore, in general, the skin comes in contact
with agglomerated NPs with a diameter bigger than 100 nm, and the presence of
acid pH and sweat can increase their tendency to aggregate. Moreover, in contact
with sweat, NPs can release metals or impurities in higher amount than bulk mate-
rial for the high ratio surface/mass. These aspects are more relevant when toxic or
sensitized metals can be released (Cd, Ni, Pd, Ag, etc.) [12].
11.3.2.3 Size
Larese Filon et al. in [12] suggested some critical sizes to evaluate NP skin hazard:
for NPs <4 nm, penetration has been demonstrated; for NPs 420 nm, skin penetra-
tion/permeation is possible, probably through follicles; for NPs 2145 nm, skin
absorption can be possible only on impaired skin; and for NPs >45 nm, skin absorp-
tion is unlikely in healthy skin.
Fig. 11.1 Metal NPs can

release more ions than bulk
metals and skin absorption
can happen as ions or as
NPs
11.3.2.4 Release of Toxic or Sensitization Substances
Due to their high surface/mass ratio, NPs can release metals in ionic form that can
penetrate the skin inducing local or systemic effects. NPs can induce in more effi-
cient way skin sensitization with respect to bulk materials. Ni, Cd, Co, and Pd NPs
can release metals that can cause allergic contact dermatitis (Fig. 11.1). Journey and
Goldman reported in 2014 [13] a case of skin and respiratory sensitization in women
exposed to NiNPs. No other case reports are available in literature.
Quantum dots containing Cd can be absorbed through the skin reaching general
blood circulation causing sign of systemic intoxication of Cd in exposed animals
[26], while no data are available in exposed workers. Polycyclic aromatic hydrocar-
bons can be released from NPs [27], and their carcinogen effect could be exerted on
the skin. Therefore, no human data are available on that.
11.3.2.5 Coating and pH
Very few data are available on this topic, but coating and pH could influence disso-
lution, release of sensitized or toxic metals, and also persistence on the skin. While
many data are available on different cells in vitro, no data are available on real work
scenario. Ovissipour et al. [28] studied decontamination of tomatoes treated with
different NPs, and he found that decontamination could be difficult from surfaces in
particular conditions of pH of the solution used and isoelectric point of NPs.
11.3.3 Skin Contamination

11.3.3.1 Area of the Skin Contaminated with NPs
Absorption of NPs through the skin is a function of surface area contaminated,

applied dose, time of contact, thickness of the membrane, and characteristics of
the NPs, On the other site, the use of protective measures to avoid skin
148 F. Larese Filon
contamination is extremely important, due to the difficulties in removal of NPs

from the skin.
11.3.3.2 Applied Dose
The amount of NPs that come in contact to the skin is important for decontamina-
tion purposes and for the loading on the skin surface. Nevertheless, in the condition
of substances with very low permeation rate, such as NPs, the dose could be not
relevant because a decontamination is not easy or possible, and NPs can be stored
in hair follicles and from there they can release ions. Moreover, NPs can penetrate
and permeate the skin if their size is less than 45 nm, as suggested by Larese Filon
et al. [12].
11.3.3.3 Time of Contact
Skin effect or absorption can be more effective when skin is exposed for longer
period. One of the crucial aspects is that after contact with NPs, it is quite difficult
to clean the skin because NPs are very adhesive to the skin and they reach the hair
follicles where they cannot be removed. In this condition also, after a short contact
with the skin, NPs cannot be removed by the cleaning procedures. There are no, in
our knowledge, studies on this topic, but there are experimental data that demon-
strated that hair follicles are the storage place for NPs that can come in contact to
the skin [29]. Moreover, one study evaluated the cleaning effect on tomatoes con-
taminated by NPs washed using deionized water. The washing was effective to
remove alumina NPs but not titania and silica [29]. The NPs persistence on the
surface was explained with differences between pH of the washing solution and NPs
isoelectric point. The Fourier transform infrared spectroscopy results showed that
some NPs can bind to certain biochemical components such as polysaccharides and
proteins on the surface of tomato skins.
In human skin, NPs can penetrate between the stratum corneum and inside the
hair follicles when NPs are flexible or when rigid NPs are smaller than 4045 nm,
particularly when the skin is impaired [12]. From hair follicles NPs can release
toxic or sensitized substances or can cross the epithelia reaching the derma. Rancan
et al. [29] demonstrated that silica NPs can reach Langerhans cells after they have
been stored in hair follicles.
11.3.3.4 Condition of the Skin Barrier
Occupational skin diseases (OSD) are extremely common in worker populations.

The European Agency for Safety and Health at Work, EU-OSHA, EU-25 report
2008 [30], reported that they constitute a top-priority public health problem. The
economic burden of OSD in the EU exceeds 5 billion spent every year on
treatment, compensation, and loss of productivity [31]. They represent up to 35 % of

all reported occupational diseases [32, 33]. The impairment of the skin is frequent
in some professions, particularly in wet workers, construction workers, health-care
workers, hairdressers, mechanics, etc. that are doing wet work or are in contact
with irritants and sensitizing agents.
The impairment of the skin barrier increases the risk of NPs penetration because
NPs can reach easily the basal layers of the epidermis and hair follicles [29]. The
impairment of the skin increases NPs penetration 4100 times [3436], and for
some NPs, such as titanium dioxide, skin penetration is possible only in impaired
skin [37]. Frequent exposure to water, irritant chemicals, detergents, disinfectants,
and powders can lead to fissuring of the skin [38]. Moreover it is possible for the
skin barrier to be compromised, although there are no visible signs [39]. In this case,
the contamination of the skin with NPs can cause an increased risk of NPs penetra-
tion into the skin and possible effects. In vitro data performed using human skin and
Franz cells demonstrated that, obviously, the impairment of the skin increases sig-
nificantly the metal content inside the skin and skin absorption for many metal NPs
[18, 34, 36, 40, 41].
11.3.4 Skin Decontamination and Cleansing Procedures
No data are available on the better way to decontaminate and to clean the skin after
contact with NPs. The possible persistence of NPs on skin surface, inside the stratum
corneum, and the storage on hair follicles suggests the need to avoid direct skin con-
tact with NPs. Decontamination of tomato surface after contact with alumina, silica,
and titania NPs can be more or less effective in function of pH of the solution used
and the isoelectric point of the NPs [28]. Moreover, cleansing procedures can enhance
penetration, increasing pH of the skin and reducing the skin barrier properties. No
data are available on workers but data on decontamination from the skin of lead pow-
ders (non-nano) in in vitro condition demonstrated that different cleaning procedures
can influence decontamination effectiveness and can increase skin absorption of lead
[42]. Considering that decontamination procedures can be less effective after the
contact with NPs, we stress the need to avoid skin contamination at all.
11.3.5 Skin Protection
Gloves made of neoprene, nitrile, or other chemical-resistant gloves should be used

and changed frequently or whenever they are visibly worn, torn, or contaminated to
protect the hands from the direct or indirect contact with NPs [1]. We suggest the
use of double gloves to increase the protection. Work apron with long trousers, long
sleeves, gauntlets, and caps are recommended to avoid contamination with other
parts of the body skin that can increase the exposed area and through skin absorp-
tion of NPs.
150 F. Larese Filon
11.4 Discussion and Conclusions
Skin exposure to NPs can be relevant for local or systemic effect in a particular con-
dition and where it is possible skin contact with NPs that can release sensitizing (i.e.,
Ni, Pd, Co) or toxic metals (i.e., Cd, As). The high rate surface/mass permits NPs to
release metallic ions in higher amount vs bulk material. Ions can penetrate the skin
and cause allergic sensitization or allergic symptoms in already sensitized subjects or
can induce allergic sensitization. Moreover, there is only one case report of sensitiza-
tion and symptoms in a woman exposed to nickel NPs [13], while only animal data
are available on cadmium presence into internal organs in rats exposed via skin to
Cd-selenite quantum dots [26]. NPs can release polyaromatic hydrocarbons (PAH)
that can have local or systemic effects, due also to their carcinogen effect [27].
For NPs that cannot release toxic/sensitizing metals or chemicals, we have two
options:
1. Soft NPs as liposomes can penetrate and permeate the skin because they can
squeeze between cells.
2. Rigid NPs: only very small NPs can penetrate and permeate the intact skin (<4
nm). For NPs 420 nm, a skin penetration/permeation is possible, probably
through follicles; for NPs 2145 nm, skin absorption can be possible only on
impaired skin; for NPs >45 nm, skin absorption is unlikely in healthy skin.
In these conditions, the presence of an impaired skin barrier in workers needs
to be evaluated, since skin absorption can happen also for bigger NPs.
Moreover, considering that NPs can persist on the skin despite decontaminate
measures, it is compulsory to avoid skin contamination using personal protective
equipment. Labeling is needed for NPs and products containing sensitizing or toxic
metals to advise users to protect them from direct contact with the skin.
Conflict of Interest None
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ISSN 2364-8333 ISSN 2364-8341 (electronic)

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Springer Science+Business Media Singapore 2017

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We welcome newcomers to our SC. Anyone who is interested in allergy and

Kurashiki, Japan Takemi Otsuki

1 Suppressive Effects of Asbestos Exposure on the Human

7 Skin Sensitization Model Based on Only Animal

Yasumitsu Nishimura, Naoko Kumagai-Takei, Megumi Maeda,

Abstract Asbestos exposure causes malignancies such as mesothelioma and lung

Keywords Asbestos NK cell CTL Th1 cell Treg Immune surveillance

Y. Nishimura N. Kumagai-Takei H. Matsuzaki S. Lee S. Yamamoto T. Hatayama

Springer Science+Business Media Singapore 2017 1

1.1 Asbestos, Malignant Mesothelioma (MM), and Immune

1.2 Alteration of Various Immune Cells Caused by Asbestos

1.2.1 Natural Killer (NK) Cell

1.2.1.2 NK Cell Cytotoxicity in MM Patients

We investigated the state of NK cell cytotoxicity in patients with MM who might

1.2.1.3 Relationship Between Killing Activity and Receptor Expressions

The relationship between expression levels of NK cell-activating receptors with the

1.2.2 Cytotoxic T Lymphocyte (CTL)

1.2.2.1 CTL Differentiation and Proliferation in Mixed Lymphocyte

1.2.2.2 Characteristics of CTL in Asbestos-Exposed Patients

CTL differentiation and proliferation were impaired by asbestos exposure in

1.2.3 Alteration of T Cell Caused by Asbestos Exposure

1.2.3.1 T Helper 1 (Th1) Cell

Asbestos Exposure on Human T Cell Line, MT-2

A human T cell line, MT-2 (human T cell leukemia/lymphoma virus 1 (HTLV-1)

CXCR3 and IFN- in Peripheral Th1 Cells Cultured with CB

CXCR3 Expression of Peripheral CD4+ Cells in MM and PP Patients

1.2.3.2 Regulatory T Cell (Treg)

In Vitro Assessment of Treg Function Exposed to Asbestos Using MT-2

The abovementioned cell line model continuously exposed to asbestos was

1.3 Summary of Asbestos-Induced Reduction of Antitumor

Table 1.1 summarizes investigation of the experimental immunological effects of

Table 1.1 Investigations of the experimental immunological effects of asbestos exposure on

Table 1.1 (continued)

and Bcl-2 in resistance against an asbestos-induced apoptosis of T cells. Apoptosis.

50. Maeda M, Nishimura Y, Kumagai N, Hayashi H, Hatayama T, Katoh M, Miyahara N,

Suni Lee, Hiroaki Hayashi, Hidenori Matsuzaki, Naoko Kumagai-Takei,

Abstract Occupational and chronic exposure to silica particles causes pulmonary

S. Lee H. Matsuzaki N. Kumagai-Takei K. Yoshitome S. Yamamoto T. Hatayama

Springer Science+Business Media Singapore 2017 15

Keywords Silica Autoimmune diseases Regulatory T cell CD95/Fas

2.1 Silica Exposure and Pulmonary Diseases

Silica-exposed patients suffer from pulmonary fibrosis known as silicosis, a typical

2.2 Immune Complications

2.3 Alteration of Immune Cells and Functions

2.3.1 Responder T Cell Activation Caused by Silica

Although it is commonly thought that immunotoxicity may kill immune-competent

2.3.2 Inhibitory Molecules for CD95/Fas-Mediated Apoptosis

immunological pathophysiological status of SIL. We will report the findings of

2.3.3 Regulatory T Cells in Silicosis Peripheral Blood

2.3.4 Autoantibodies in SIL

9. Pelucchi C, Pira E, Piolatto G, Coggiola M, Carta P, La Vecchia C. Occupational silica expo-

32. Wu P, Hyodoh F, Hatayama T, Sakaguchi H, Hatada S, Miura Y, Takata-Tomokuni A,

66. Ueki A, Isozaki Y, Tomokuni A, Tanaka S, Otsuki T, Kishimoto T, Kusaka M, Aikoh T,

Claudia Petrarca, Luca Di Giampaolo, Paola Pedata, Sara Cortese,

Springer Science+Business Media Singapore 2017 27

experimental investigations in cellular and animal models. Moreover, first emerging

E. Kuroda () K.J. Ishii ()